BISPECIFIC-Fc MOLECULES

ABSTRACT

Described herein is a bispecific molecule containing an Fc polypeptide chain and immunoglobulin variable regions. Also provided are pharmaceutical formulations comprising such molecules, nucleic acids encoding such molecules, host cells containing such nucleic acids, methods of making such molecules, and methods of using such molecules.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application61/791,424, filed Mar. 15, 2013, the content of which is incorporatedherein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 11, 2014, isnamed A-1810-US-NP_SL.txt and is 58,831 bytes in size.

BACKGROUND

Bispecific antibodies have promise as therapeutics in a variety ofindications. Bispecific antibodies having a standard IgG format can bechallenging to produce because they include four different polypeptidechains. The efficacy of a smaller, more easily-produced bispecificmolecule has been clinically demonstrated in non-Hodgkin's lymphoma.See, e.g., Bargou et al. (2008), Science 321(5891): 974-977. Dailyadministration was used to achieve these results, presumably because ofthe short in vivo half life of this small, single chain molecule. Id.Hence, there is a need in the art for bispecific therapeutics withfavorable pharmacokinetic properties, as well as therapeutic efficacyand a format that makes them straightforward to produce.

SUMMARY

A Bispecific-Fc (Bi-Fc) as described herein molecule can bind to onemolecule of each of two different proteins and contains an Fc region ofan antibody. A Bi-Fc also can have favorable pharmacokinetic propertiesrelative to a single chain molecule lacking an Fc region. One proteinbound by a Bi-Fc can be expressed on an immune effector cell such as a Tcell, an NK cell, a neutrophil, or a macrophage, and the other proteincan be expressed on a target cell, for example, a cancer cell, a cellinfected by a pathogen, or a cell mediating a disease, such as afibrotic cell. The Bi-Fc molecules described herein can elicitactivation an immune effector cell in the presence of a target cell.

In one aspect, provided herein is a Bi-Fc, which can comprise: (a) (i) afirst polypeptide chain having the formula V1-L1-V2-L2-V3-L3-V4-L4-Fc,wherein Fc is an Fc polypeptide chain, wherein V1, V2, V3, and V4 areeach immunoglobulin variable regions that have different amino acidsequences, wherein L1, L2, L3, and L4 are linkers, and wherein L4 can bepresent or absent, and (ii) a second polypeptide chain that comprises anFc polypeptide chain; or (b) (i) a first polypeptide chain having theformula Fc-L4-V1-L1-V2-L2-V3-L3-V4, wherein Fc is an Fc polypeptidechain, wherein V1, V2, V3, and V4 are each immunoglobulin variableregions that have different amino acid sequences, wherein L1, L2, L3,and L4 are linkers, and wherein L4 can be present or absent, and (ii) asecond polypeptide chain that comprises an Fc polypeptide chain; whereinthe Bi-Fc mediates cytolysis of a target cell displaying a target cellprotein by an immune effector cell, and does not mediate cytolysis of acell not displaying the target cell protein by the immune effector celland/or wherein the Bi-Fc can bind to a target cell and to an immuneeffector cell. V1 can be a heavy chain variable (VH) region, and V2 canbe a light chain variable (VL) region. In an alternate embodiment, V1can be a VL region and V2 can be a VH region. V3 and V4 can be a VH anda VL region, respectively, or V3 and V4 can be a VL and a VH region,respectively. L1 and L3 can be at least 15 amino acids long, and L2 canbe less than 12 amino acids long. V1 and V2 can bind to a target cell oran immune effector cell when they are part of an IgG and/or an scFvantibody, and V3 and V4 can bind to a target cell or an immune effectorcell when they are part of an IgG and/or an scFv antibody. The Fcpolypeptide chain in the first polypeptide chain can comprise aheterodimerizing alteration, and the Fc polypeptide chain in the secondpolypeptide chain can comprise another heterodimerizing alteration. Theheterodimerizing alteration in the first polypeptide chain can be acharge pair substitution, and the heterodimerizing alteration in thesecond polypeptide chain can be a charge pair substitution. The firstpolypeptide chain can comprise the charge pair substitutions K409D orK409E and K392D or K392E, and the second polypeptide chain can comprisethe charge pair substitutions D399K or D399R and D356K or D356R; or thesecond polypeptide chain comprises the charge pair substitutions K409Dor K409E and K392D or K392E, and the first polypeptide chain comprisesthe charge pair substitutions D399K or D399R and D356K or D356R. The Fcpolypeptide chains of the first and second polypeptide chains can behuman IgG Fc polypeptide chains, such as IgG1, IgG2, IgG3, or IgG4 Fcpolypeptide chains. The Fc polypeptide chains of the first and secondpolypeptide chains can comprise one or more alterations that inhibit(s)Fc gamma receptor (FcγR) binding or enhance(s) ADCC. The Fc polypeptidechains of the first and second polypeptide chains comprise, for example,L234A, L235A, and any substitution at N297.

In a further aspect, described herein is a Bi-Fc, which can comprise:(i) a first polypeptide chain having following formula:V1-L1-V2-L2-V3-L3-V4-L4-Fc, wherein Fc is an Fc polypeptide chain,wherein V1, V2, V3, and V4 are each immunoglobulin variable regions thathave different amino acid sequences, wherein L1, L2, L3, and L4 arelinkers, and wherein L4 can be present or absent; and (ii) a secondpolypeptide chain comprising an Fc polypeptide chain; wherein L1 and L3are at least 15 amino acids long and L2 is less than 12 amino acidslong; wherein either V1 is a VH region and V2 is a VL region or V1 is aVL region and V2 is a VH region; wherein either V3 is a VH region and V4is a VL region or V3 is a VL region and V4 is a VH region; wherein theFc polypeptide chains of each of the first and second polypeptide chainseach contain a heterodimerizing alteration; and wherein the Bi-Fcmediates cytolysis of a target cell displaying a target cell protein byan immune effector cell, and does not mediate cytolysis of a cell notdisplaying the target cell protein by the immune effector cell, and/orthe Bi-Fc can bind to a target cell and to an immune effector cell. TheFc polypeptide chains can be human IgG Fc polypeptide chains, such asIgG1, IgG2, IgG3, or IgG4 Fc polypeptide chains. The Fc polypeptidechains of the first and second polypeptide chains comprise one or morealteration that inhibits FcγR binding, such as one or more of L234A,L235A, and any substitution at N297.

In a further aspect, a Bi-Fc can comprise: (a) a first polypeptide chainhaving the formula V1-L1-V2-L2-V3-L3-V4-L4-Fc, wherein Fc is an Fcpolypeptide chain, wherein V1, V2, V3, and V4 are each immunoglobulinvariable regions that have different amino acid sequences, wherein L1,L2, L3, and L4 are linkers, and wherein L4 can be present or absent; or(b) a first polypeptide chain having the following formula:Fc-L4-V1-L1-V2-L2-V3-L3-V4, wherein Fc is an Fc polypeptide chain,wherein V1, V2, V3, and V4 are each immunoglobulin variable regions thathave different amino acid sequences, wherein L1, L2, L3, and L4 arelinkers, and wherein L4 can be present or absent; wherein the Bi-Fc is amonomer and wherein the Bi-Fc mediates cytolysis of a target celldisplaying a target cell protein by an immune effector cell, and doesnot mediate cytolysis of a cell not displaying the target cell proteinby the immune effector cell, and/or the Bi-Fc can bind to a target celland to an immune effector cell. The Fc polypeptide chain can be a humanIgG Fc polypeptide chain, such as IgG1, IgG2, IgG3, or IgG4 Fcpolypeptide chain. The Fc polypeptide chain of (a) or (b) can compriseone or more the following alterations: K392D, K382E, K409D, K409E,Y349T, L351T, L368T, L398T, F405T, Y407T, Y407R. The Fc polypeptidechain of (a) or (b) can comprise one or more alteration that inhibitsFcγR binding, such as one or more of L234A, L235A, and any substitutionat N297.

The immune effector cell of any Bi-Fc described herein can be a human Tcell and/or a cynomolgus monkey T cell. The effector cell protein of anyBi-Fc described herein can be part of the human and/or cynomolgus monkeyTCR-CD3 complex. The effector cell protein of any Bi-Fc described hereincan be the human and/or cynomolgus monkey TCRα, TCRβ, TCRγ, TCRδ, CD3βchain, CD3γ chain, CD3δ chain, CD3ε chain, or CD3ζ chain.

If the effector cell protein is the CD3ε chain, the Bi-Fc can comprise aVH region and a VL comprising the amino acid sequences of SEQ ID NOs:7and 8, respectively, or comprising the amino acid sequences of SEQ IDNOs:29 and 31, respectively.

The target cell of any Bi-Fc can be a cancer cell, a cell infected by apathogen, or a cell that mediates disease. If target cell is a cancercell, the cancer can be a hematologic malignancy or a solid tumormalignancy. If the target cell is a cell infected by a pathogen, thepathogen can be virus, including human immunodeficiency virus, hepatitisvirus, human papilloma virus, or cytomegalovirus, or a bacterium of thegenus Listeria, Mycobacterium, Staphylococcus, or Streptococcus. If thetarget cell is a cell that mediates a disease, the target cell can be afibrotic cell that mediates a fibrotic disease or an autoimmune orinflammatory disease.

Provided herein is pharmaceutical formulation comprising any of theBi-Fc molecules described herein and a physiologically acceptableexcipient.

Further provided herein are nucleic acids encoding any of the Bi-Fcdescribed herein and vectors containing such nucleic acids, as well ashost cell containing such nucleic acids and/or vectors. In anotheraspect, described herein is a method for making a Bi-Fc comprisingculturing the host cell containing the nucleic acids or vector underconditions such that the nucleic acids are expressed, and recovering theBi-Fc from the cell mass or the culture medium.

In another aspect, provided herein is a method for treating a cancerpatient comprising administering to the patient a therapeuticallyeffective dose of any of the Bi-Fc molecules described herein, whereinthe target cell of the Bi-Fc is a cancer cell. This method can furthercomprise administering radiation, a chemotherapeutic agent, or anon-chemotherapeutic, anti-neoplastic agent before, after, orconcurrently with the administration of the Bi-Fc. The patient can havea hematologic malignancy or a solid tumor malignancy.

In a further embodiment, described herein is a method for treating apatient having a fibrotic disease comprising administering to thepatient a therapeutically effective dose of any of the Bi-Fc moleculesdescribed herein, wherein the target cell of the Bi-Fc is a fibroticcell. The fibrotic disease can be atherosclerosis, chronic obstructivepulmonary disease (COPD), cirrhosis, scleroderma, kidney transplantfibrosis, kidney allograft nephropathy, or a pulmonary fibrosis,including idiopathic pulmonary fibrosis.

In still another aspect, described herein is a method for treating apatient having a disease mediated by a pathogen comprising administeringto the patient a therapeutically effective dose of any of the Bi-Fcmolecules described herein. The pathogen can be a virus, a bacterium, ora protozoan.

Also provided herein is a pharmaceutical composition comprising any ofthe Bi-Fc molecules described herein. Such compositions can be for thetreatment of a cancer, an infectious disease, an autoimmune orinflammatory disease, or a fibrotic disease.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: A diagram of a Bi-Fc molecule. Four immunoglobulin variableregions are indicated by ovals and labeled V1, V2, V3, and V4. CH2 andCH3 regions are labeled as such and diagramed as an elongated hexagon.Lines between these regions indicate linkers or a hinge region.Exemplary disulfide bridges are indicated by horizontal lines.

FIG. 2: Binding of a Bi-Fc to target cell and immune effector cells.Methods are described in Example 2. Mean fluorescence intensity (MFI) isindicated on the x axis, and the number of cells is indicated on the yaxis. The unfilled profiles represent data from cells in the absence ofone of the bispecific molecules, and the solidly filled profilesrepresent data from cells in the presence of one of the bispecificmolecules. As indicated in the figure, panels at left represent datafrom samples containing the anti-HER2/CD3 Bi-Fc, and panels at rightrepresent data from samples containing the single chain anti-HER2/CD3.Top two panels represent data from samples containing JIMT-1 cells(which express the target cell protein HER2), and bottom two panelsrepresent data from samples containing T cells (which express theeffector cell protein CD3ε).

FIG. 3: Cytolytic activity of an anti-FOLR1/CD3 Bi-Fc and a single chainanti-FOLR1/CD3 molecule. Methods are described in Example 3. The x axisin each panel indicates the concentration of the Bi-Fc or single chainmolecule (pM) in each sample. The y axis in each panel indicates thepercent specific lysis calculated as described in Example 3. Opencircles connected by a dashed line indicate data from samples containingthe single chain molecule, and filled circles connected by a solid lineindicate data from the Bi-Fc molecule. The top, middle, and bottompanels, as indicated, show data from Cal-51 cells (which express FOLR1),T47D cells (which express FOLR1), and BT474 cells (which do not expressFOLR1), respectively.

FIG. 4: Cytolytic activity of an anti-HER2/CD3 Bi-Fc and a single chainanti-HER2/CD3 molecule. Methods are described in Example 3. The x axisin each panel indicates the concentration of the Bi-Fc or single chainmolecule (pM) in each sample. The y axis in each panel indicates thepercent specific lysis calculated as described in Example 3. Opencircles connected by a dashed line indicate data from samples containingthe single chain molecule, and filled circles connected by a solid lineindicate data from the Bi-Fc molecule. The top, middle, and bottompanels, as indicated, show data from JIMT-1 cells (which express HER2),T47D cells (which express HER2), and SHP77 cells (which do not expressHER2), respectively.

FIG. 5: Cytokine production by T cells in the presence of ananti-FOLR1/CD3 Bi-Fc or single chain molecule. Methods are described inExample 4. Open circles connected by dashed lines indicate data fromassays containing the anti-FOLR1/CD3 Bi-Fc, and solidly filled circlesconnected by solid lines indicate data from the single chainanti-FOLR1/CD3 molecule. The x axis in each panel indicates theconcentration of the Bi-Fc or single chain molecule (pM) in each assay.The y axis indicates the concentration and identity of the cytokinedetected (pg/mL). Panel A shows data for interferon gamma (IFNγ). PanelB shows data for tumor necrosis factor alpha (TNFα). Panel C shows datafor interleukin-10 (IL-10). Panel D shows data for interleukin-2 (IL-2).Panel E shows data for interleukin-13 (IL-13). As indicated, graphs onthe left show data from T47D cells (which express FOLR1), and graphs onthe right show data from BT474 cells (which do not express FOLR1).

FIG. 6: Cytokine production by T cells in the presence of ananti-HER2/CD3 Bi-Fc or single chain molecule. Methods are described inExample 4. Open circles connected by dashed lines indicate data fromassays containing the anti-HER2/CD3 Bi-Fc, and solidly filled circlesconnected by solid lines indicate data from the single chainanti-HER2/CD3 molecule. The x axis in each panel indicates theconcentration of the Bi-Fc or single chain molecule (pM) in each assay.The y axis indicates the concentration and identity of the cytokinedetected (pg/mL). Panels A, B, C, D, and E show data for IFNγ, TNFα,IL-10 IL-2, and IL-13, respectively, as indicated. As indicated, panelson the left show data from JIMT-1 cells (which express HER2), and panelson the right show data from SHP77 cells (which do not express HER2).

FIG. 7: Percentage of CD25⁺ and CD69⁺ cells in the presence of ananti-HER2/CD3 Bi-Fc or single chain molecule. Methods are described inExample 5. The x axis indicates the concentration (pM) of theanti-HER2/CD3 Bi-Fc or single chain molecule. The y axis indicates thepercent of CD3⁺ T cells that are also CD25⁺ (left panel) or CD69⁺ (rightpanel) cells. Symbols indicate as follows: open squares connected bydashed line, the single chain molecule plus JIMT-1 target cells; solidlyfilled, downward pointing triangles connected by a solid line, the Bi-Fcmolecule plus JIMT-1 target cells; open circles connected by a dashedline, the single chain molecule without JIMT-1 target cells; and solidlyfilled, upward pointing triangles connected by a solid line, the Bi-Fcwithout JIMT-1 target cells.

FIG. 8: Pharmacokinetic properties of a Bi-Fc and a single chainbispecific molecule in mice. Methods are described in Example 6. In thetop panel, a pharmacokinetic profile following an intravenous injectionis shown, and below is shown the profile following a subcutaneousinjection. Solidly filled circles connected by a solid line indicatedata from the anti-HER2/CD3 single chain molecule, and asterisksconnected by a solid line indicate data from the anti-HER2/CD3 Bi-Fcmolecule.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO Description SEQ ID NO: 1 Amino acid sequence preceding VH CDR1SEQ ID NO: 2 Amino acid sequence preceding VH CDR2 SEQ ID NO: 3 Aminoacid sequence following VH CDR3 SEQ ID NO: 4 Amino acid sequencefollowing light chain CDR3 SEQ ID NO: 5 Amino acid sequence of anti-HER2VH region SEQ ID NO: 6 Amino acid sequence of anit-HER2 VL region SEQ IDNO: 7 Amino acid sequence of anti CD3ε VH region SEQ ID NO: 8 Amino acidsequence of anti-CD3ε VL region SEQ ID NO: 9 Amino acid sequence of asingle chain anti-HER2/CD3 (P136629.3) SEQ ID NO: 10 Amino acid sequenceof a first polypeptide chain of an anti- HER2/CD3 of a Bi-Fc SEQ ID NO:11 Nucleic acid sequence of SEQ ID NO: 10 SEQ ID NO: 12 Amino acidsequence of a human IgG1 Fc polypeptide containing alterations D356K andD399K SEQ ID NO: 13 Nucleic acid sequence encoding SEQ ID NO: 12 SEQ IDNO: 14 Amino acid sequence of ao single chain anti-FOLR1/CD3 moleculeSEQ ID NO: 15 Amino acid sequence of a first polypeptide chain of ananti- FOLR1/CD3 molecule SEQ ID NO: 16 Nucleic acid sequence encodingSEQ ID NO: 15 SEQ ID NO: 17 Amino acid sequence of a linker SEQ ID NO:18 Amino acid sequence of a linker SEQ ID NO: 19 Amino acid sequence ofa linker SEQ ID NO: 20 Amino acid sequence of a linker SEQ ID NO: 21Amino acid sequence of a linker SEQ ID NO: 22 Mature amino acid sequenceof CD3 epsilon chain of Homo sapiens SEQ ID NO: 23 Mature amino acidsequence of CD3 epsilon chain of Macaca fascicularis SEQ ID NO: 24 Aportion of an epitope that is part of CD3 epsilon SEQ ID NO: 25 Aminoacid sequence of human IgG1 Fc region SEQ ID NO: 26 Amino acid sequenceof human IgG2 Fc region SEQ ID NO: 27 Amino acid sequence of human IgG3Fc region SEQ ID NO: 28 Amino acid sequence of human IgG4 Fc region SEQID NO: 29 Amino acid sequence of an anti-CD3ε VH region SEQ ID NO: 30Nucleic acid sequence encoding SEQ ID NO: 29 SEQ ID NO: 31 Amino acidsequence of an anti-CD3ε VL region SEQ ID NO: 32 Nucleic acid sequenceencoding SEQ ID NO: 31

DETAILED DESCRIPTION

Described is a new form of bispecific antibody, called herein a Bi-Fc,which contains one polypeptide chain or two different polypeptidechains. One chain comprises two heavy chain variable (VH) regions, twolight chain variable (VL) regions, and an Fc polypeptide chain, and anoptional second polypeptide chain comprises an Fc polypeptide chain. Insome embodiments, one of the proteins to which the Bi-Fc binds isexpressed on the surface of an immune effector cell, such as a T cell,an NK cell, a macrophage, or a neutrophils, and the other protein towhich the Bi-Fc binds is expressed on the surface of a target cell, forexample a cancer cell, a cell infected by a pathogen, or a cell thatmediates a disease, such as, for example, a fibrotic disease. Since aBi-Fc has only one binding site for each of these proteins (i.e., itbinds each protein “monovalently,” as meant herein), its binding, byitself, will not oligomerize the proteins it binds to on a cell surface.For example, if it binds to CD3 on the surface of a T cell, CD3 will notbe oligomerized on the T cell surface in the absence of a target cell.Oligomerization of CD3 can cause a generalized activation of a T cell,which can be undesirable. The Bi-Fc tethers an immune effector cell to atarget cell, thereby eliciting specific cytolytic activity against thetarget cell, rather than a generalized inflammatory response. Further,the Bi-Fc molecules have favorable pharmacokinetic properties and arenot unduly complex to manufacture since they contain only one or onlytwo different polypeptide chains.

DEFINITIONS

An “antibody,” as meant herein, is a protein containing at least one VHor VL region, in many cases a heavy and a light chain variable region.Thus, the term “antibody” encompasses molecules having a variety offormats, including single chain Fv antibodies (scFv, which contain VHand VL regions joined by a linker), Fab, F(ab)₂′, Fab′, scFv:Fcantibodies (as described in Carayannopoulos and Capra, Ch. 9 inFUNDAMENTAL IMMUNOLOGY, 3^(rd) ed., Paul, ed., Raven Press, New York,1993, pp. 284-286) or full length antibodies containing two full lengthheavy and two full length light chains, such as naturally-occurring IgGantibodies found in mammals. Id Such IgG antibodies can be of the IgG1,IgG2, IgG3, or IgG4 isotype and can be human antibodies. The portions ofCarayannopoulos and Capra that describe the structure of antibodies areincorporated herein by reference. Further, the term “antibody” includesdimeric antibodies containing two heavy chains and no light chains suchas the naturally-occurring antibodies found in camels and otherdromedary species and sharks. See, e.g., Muldermans et al., 2001, J.Biotechnol. 74:277-302; Desmyter et al., 2001, J. Biol. Chem.276:26285-90; Streltsov et al. (2005), Protein Science 14: 2901-2909. Anantibody can be “monospecific” (that is, binding to only one kind ofantigen), “bispecific” (that is, binding to two different antigens), or“multispecific” (that is, binding to more than one different antigen).Further, an antibody can be monovalent, bivalent, or multivalent,meaning that it can bind to one, two, or multiple antigen molecules atonce, respectively. An antibody binds “monovalent,” to a particularprotein when one molecule of the antibody binds to only one molecule ofthe protein, even though the antibody may also bind to a differentprotein as well. That is, an antibody binds “monovalently,” as meantherein, to two different proteins when it binds to only one molecule ofeach protein. Such an antibody is “bispecific” and binds to each of twodifferent proteins “monovalently.” An antibody can be “monomeric,” i.e.,comprising a single polypeptide chain. An antibody can comprise multiplepolypeptide chains (“multimeric”) or can comprise two (“dimeric”), three(“trimeric”), or four (“tetrameric”) polypeptide chains. If multimeric,an antibody can be a homomulitmer, i.e., containing more than onemolecule of only one kind of polypeptide chain, including homodimers,homotrimer, or homotetramers. Alternatively, a multimeric antibody canbe a heteromultimer, i.e., containing more than one different kind ofpolypeptide chain, including heterodimers, heterotrimers, orheterotetramers. An antibody can have a variety of possible formatsincluding, for example, monospecific monovalent antibodies (as describedin International Application WO 2009/089004 and US Publication2007/0105199, the relevant portions of which are incorporated herein byreference) that may inhibit or activate the molecule to which they bind,bivalent monospecific or bispecific dimeric Fv-Fc, scFv-Fc, or diabodyFc, monospecific monovalent scFv-Fc/Fc's, the multispecific bindingproteins and dual variable domain immunoglobulins described in USPublication 2009/0311253 (the relevant portions of which areincorporated herein by reference), the heterodimeric bispecificantibodies described herein, and the many formats for bispecificantibodies described in Chapters 1, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14of BISPECIFIC ANTIBODIES, Kontermann, ed., Springer, 2011 (whichchapters are incorporated herein by reference), among many otherpossible antibody formats.

A “Bi-Fc,” as meant herein, comprises a first polypeptide chain and,optionally, a second polypeptide chain. In many embodiments, a Bi-Fccomprises both a first and a second polypeptide chain. In someembodiments, a Bi-Fc is a monomer comprising only the first polypeptidechain. The first polypeptide chain comprises two VH regions and two VLregions separated by linkers and an Fc polypeptide chain. The Fcpolypeptide chain can be N-terminal or C-terminal relative to the fourimmunoglobulin variable regions, and it can be joined to the variableregions via a linker. This linker can be present or absent. The secondpolypeptide chain, if present, comprises an Fc polypeptide chain. Thus,a Bi-Fc can be a monomer or a heterodimer. A Bi-Fc can bind to an immuneeffector cell via an effector cell protein and to a target cell via atarget cell protein and can mediate cytolysis of a target cell by animmune effector cell.

A “cancer cell antigen,” as meant herein, is a protein expressed on thesurface of a cancer cell. Some cancer cell antigens are also expressedon some normal cells, and some are specific to cancer cells. Cancer cellantigens can be highly expressed on the surface of a cancer cell. Thereare a wide variety of cancer cell antigens. Examples of cancer cellantigens include, without limitation, the following human proteins:epidermal growth factor receptor (EGFR), EGFRvIII (a mutant form ofEGFR), melanoma-associated chondroitin sulfate proteoglycan (MCSP),mesothelin (MSLN), folate receptor 1 (FOLR1), and human epidermal growthfactor 2 (HER2), among many others.

“Chemotherapy,” as used herein, means the treatment of a cancer patientwith a “chemotherapeutic agent” that has cytotoxic or cytostatic effectson cancer cells. A “chemotherapeutic agent” specifically targets cellsengaged in cell division and not cells that are not engaged in celldivision. Chemotherapeutic agents directly interfere with processes thatare intimately tied to cell division such as, for example, DNAreplication, RNA synthesis, protein synthesis, the assembly,disassembly, or function of the mitotic spindle, and/or the synthesis orstability of molecules that play a role in these processes, such asnucleotides or amino acids. A chemotherapeutic agent therefore hascytotoxic or cytostatic effects on both cancer cells and other cellsthat are engaged in cell division. Chemotherapeutic agents arewell-known in the art and include, for example: alkylating agents (e.g.busulfan, temozolomide, cyclophosphamide, lomustine (CCNU),methyllomustine, streptozotocin, cis-diamminedi-chloroplatinum,aziridinylbenzo-quinone, and thiotepa); inorganic ions (e.g. cisplatinand carboplatin); nitrogen mustards (e.g. melphalan hydrochloride,ifosfamide, chlorambucil, and mechlorethamine HCl); nitrosoureas (e.g.carmustine (BCNU)); anti-neoplastic antibiotics (e.g. adriamycin(doxorubicin), daunomycin, mitomycin C, daunorubicin, idarubicin,mithramycin, and bleomycin); plant derivatives (e.g. vincristine,vinblastine, vinorelbine, paclitaxel, docetaxel, vindesine, VP-16, andVM-26); antimetabolites (e.g. methotrexate with or without leucovorin,5-fluorouracil with or without leucovorin, 5-fluorodeoxyuridine,6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea,deoxycoformycin, gemcitabine, and fludarabine); podophyllotoxins (e.g.etoposide, irinotecan, and topotecan); as well as actinomycin D,dacarbazine (DTIC), mAMSA, procarbazine, hexamethylmelamine,pentamethylmelamine, L-asparaginase, and mitoxantrone, among many knownin the art. See e.g. Cancer: Principles and Practice of Oncology, 4^(th)Edition, DeVita et al., eds., J.B. Lippincott Co., Philadelphia, Pa.(1993), the relevant portions of which are incorporated herein byreference. Alkylating agents and nitrogen mustard act by alkylating DNA,which restricts uncoiling and replication of strands. Methotrexate,cytarabine, 6-mercaptopurine, 5-fluorouracil, and gemcitabine interferewith nucleotide synthesis. Plant derivatives such a paclitaxel andvinblastine are mitotic spindle poisons. The podophyllotoxins inhibittopoisomerases, thus interfering with DNA replication. Antibioticsdoxorubicin, bleomycin, and mitomycin interfere with DNA synthesis byintercalating between the bases of DNA (inhibiting uncoiling), causingstrand breakage, and alkylating DNA, respectively. Other mechanisms ofaction include carbamoylation of amino acids (lomustine, carmustine),and depletion of asparagine pools (asparaginase). Merck Manual ofDiagnosis and Therapy, 17^(th) Edition, Section 11, Hematology andOncology, 144. Principles of Cancer Therapy, Table 144-2 (1999).Specifically included among chemotherapeutic agents are those thatdirectly affect the same cellular processes that are directly affectedby the chemotherapeutic agents listed above.

A drug or treatment is “concurrently” administered with a Bi-Fc if it isadministered in the same general time frame as the Bi-Fc, optionally, onan ongoing basis. For example, if a patient is taking Drug A once a weekon an ongoing basis and a Bi-Fc once every six months on an ongoingbasis, Drug A and the Bi-Fc are concurrently administered, whether ornot they are ever administered on the same day. Similarly, if the Bi-Fcis taken once per week on an ongoing basis and Drug A is administeredonly once or a few times on a daily basis, Drug A and the Bi-Fc areconcurrently administered as meant herein. Similarly, if both Drug A andthe Bi-Fc are administered for short periods of time either once ormultiple times within a one month period, they are administeredconcurrently as meant herein as long as both drugs are administeredwithin the same month.

A “conservative amino acid substitution,” as meant herein, is asubstitution of an amino acid with another amino acid with similarproperties. Properties considered include chemical properties such ascharge and hydrophobicity. Table 1 below lists substitutions for eachamino acid that are considered to be conservative substitutions as meantherein.

TABLE 1 Conservative Amino Acid Substitutions Original ResidueConservative Substitutions Ala Val, Leu, Ile Arg Lys, Gln, Asn Asn GlnAsp Glu Cys Ser, Ala Gln Asn Glu Asp Gly Pro, Ala His Asn, Gln, Lys, ArgIle Leu, Val, Met, Ala, Phe, Norleucine Leu Norleucine, Ile, Val, Met,Ala, Phe Lys Arg, Gln, Asn Met Leu, Phe, Ile Phe Leu, Val, Ile, Ala, TyrPro Ala Ser Thr, Ala, Cys Thr Ser Trp Tyr, Phe Tyr Trp, Phe, Thr, SerVal Ile, Met, Leu, Phe, Ala, Norleucine

As meant herein, an “Fc region” is a dimer consisting of two polypeptidechains joined by one or more disulfide bonds, each chain comprising partor all of a hinge domain plus a CH2 and a CH3 domain. Each of thepolypeptide chains is referred to as an “Fc polypeptide chain.” Todistinguish the two Fc polypeptide chains, in some instances one isreferred to herein as an “A chain” and the other is referred to as a “Bchain.” More specifically, the Fc regions contemplated for use with thepresent invention are IgG Fc regions, which can be mammalian, forexample human, IgG1, IgG2, IgG3, or IgG4 Fc regions. Among human IgG1 Fcregions, at least two allelic types are known. In other embodiments, theamino acid sequences of the two Fc polypeptide chains can vary fromthose of a mammalian Fc polypeptide by no more than 10 amino acidsubstitutions, insertions, and/or deletions of a single amino acid per100 amino acids relative to the sequence of a mammalian Fc polypeptideamino acid sequence. In some embodiments, such variations can be“heterodimerizing alterations” that facilitate the formation ofheterodimers over homodimers, an Fc alteration that extends half life,an alteration that inhibits Fc gamma receptor (FcγR) binding, and/or analteration that enhances ADCC.

An “Fc alteration that extends half life,” as meant herein is analteration within an Fc polypeptide chain that lengthens the in vivohalf life of a protein that contains the altered Fc polypeptide chain ascompared to the half life of a similar protein containing the same Fcpolypeptide, except that it does not contain the alteration. Suchalterations can be included in an Fc polypeptide chain that is part of aBi-Fc. The alterations M252Y, S254T, and T256E (methionine at position252 changed to tyrosine; serine at position 254 changed to threonine;and threonine at position 256 changed to glutamic acid; numberingaccording to EU numbering as shown in Table 2) are Fc alterations thatextend half life and can be used together, separately or in anycombination. These alterations and a number of others are described indetail in U.S. Pat. No. 7,083,784. The portions of U.S. Pat. No.7,083,784 that describe such alterations are incorporated herein byreference. Similarly, M428L and N434S are Fc alterations that extendhalf life and can be used together, separately or in any combination.These alterations and a number of others are described in detail in U.S.Patent Application Publication 2010/0234575 and U.S. Pat. No. 7,670,600.The portions of U.S. Patent Application Publication 2010/0234575 andU.S. Pat. No. 7,670,600 that describe such alterations are incorporatedherein by reference. In addition, any substitution at one of thefollowing sites can be considered an Fc alteration that extends halflife as meant here: 250, 251, 252, 259, 307, 308, 332, 378, 380, 428,430, 434, 436. Each of these alterations or combinations of thesealterations can be used to extend the half life of a heterodimericbispecific antibody as described herein. Other alterations that can beused to extend half life are described in detail in InternationalApplication PCT/US2012/070146 filed Dec. 17, 2012. The portions of thisapplication that describe such alterations are incorporated herein byreference. Some specific embodiments described in this applicationinclude insertions between positions 384 and 385 (EU numbering as shownin Table 2) that extend half life, including the following amino acidsequences: GGCVFNMFNCGG (SEQ ID NO:33), GGCHLPFAVCGG (SEQ ID NO:34),GGCGHEYMWCGG (SEQ ID NO:35), GGCWPLQDYCGG(SEQ ID NO:36), GGCMQMNKWCGG(SEQ ID NO:37), GGCDGRTKYCGG (SEQ ID NO:38), GGCALYPTNCGG (SEQ IDNO:39), GGCGKHWHQCGG (SEQ ID NO:40), GGCHSFKHFCGG (SEQ ID NO:41),GGCQGMWTWCGG (SEQ ID NO:42), GGCAQQWHHEYCGG (SEQ ID NO:43), andGGCERFHHACGG (SEQ ID NO:44), among others. Heterodimeric bispecificantibodies containing such insertions are contemplated.

An “Fc alteration that is unfavorable to homodimer formation,” includesany alteration in an Fc polypeptide chain such that the Fc polypeptidechain has decreased ability to form homodimers compared to a wild typeFc polypeptide chain. Such alterations are described in detail in U.S.Patent Application Publication US2012/0244578. The portions of thispublication that described such alteration are incorporated herein byreference. Such alterations can be included in an Fc polypeptide chainthat is part of a Bi-Fc, especially in embodiments where the Bi-Fc is amonomer. In some embodiments, such alterations occur in the CH3 regionof the Fc polypeptide chain and comprise an alteration such that one ormore charged amino acids in the wild type amino acid sequence arereplaced with amino acids electrostatically unfavorable to CH3 homodimerformation, and/or one or more hydrophobic interface residues arereplaced with a small polar amino acid, such as, for example,asparagine, cysteine, glutamine, serine, or threonine. Morespecifically, for example, a charged amino acid, e.g., lysine atposition 392 and/or position 409, can be replaced with a neutral oroppositely charged amino acid, for example aspartate or glutamate. Thiscan also occur at any other charged amino acid within the Fc polypeptidechain. Alternatively or in addition, one or more hydrophobic interfaceresidues selected from the group consisting of Y349, L351, L368, V397,L398, F405, and Y407 can be replaced with a small polar amino acid.Further, the Fc polypeptide chain can have one or more mutated cysteineresidues to prevent di-sulfide bond formation. Particularly usefulcysteine mutations in this regard are those in the hinge region of theFc polypeptide chain. Such cysteines can be deleted or substituted withother amino acids.

“Heterodimerizing alterations” generally refer to alterations in the Aand B chains of an Fc region that facilitate the formation ofheterodimeric Fc regions, that is, Fc regions in which the A chain andthe B chain of the Fc region do not have identical amino acid sequences.Such alterations can be included in an Fc polypeptide chain that is partof a Bi-Fc. Heterodimerizing alterations can be asymmetric, that is, anA chain having a certain alteration can pair with a B chain having adifferent alteration. These alterations facilitate heterodimerizationand disfavor homodimerization. Whether hetero- or homo-dimers haveformed can be assessed by size differences as determined bypolyacrylamide gel electrophoresis in some situations or by otherappropriate means (such as molecular tags or binding by antibodies thatrecognize certain portions of the heterodimer) in situations where sizeis not a distinguishing characteristic. One example of such pairedheterodimerizing alterations are the so-called “knobs and holes”substitutions. See, e.g., U.S. Pat. No. 7,695,936 and US PatentApplication Publication 2003/0078385, the portions of which describesuch mutations are incorporated herein by reference. As meant herein, anFc region that contains one pair of knobs and holes substitutions,contains one substitution in the A chain and another in the B chain. Forexample, the following knobs and holes substitutions in the A and Bchains of an IgG1 Fc region have been found to increase heterodimerformation as compared with that found with unmodified A and B chains: 1)Y407T in one chain and T366Y in the other; 2) Y407A in one chain andT366W in the other; 3) F405A in one chain and T394W in the other; 4)F405W in one chain and T394S in the other; 5) Y407T in one chain andT366Y in the other; 6) T366Y and F405A in one chain and T394W and Y407Tin the other; 7) T366W and F405W in one chain and T394S and Y407A in theother; 8) F405W and Y407A in one chain and T366W and T394S in the other;and 9) T366W in one polypeptide of the Fc and T366S, L368A, and Y407V inthe other. This way of notating mutations can be explained as follows.The amino acid (using the one letter code) normally present at a givenposition in the CH3 region using the EU numbering system (which ispresented in Edelman et al. (1969), Proc. Natl. Acad. Sci. 63: 78-85;see also Table 2 below) is followed by the EU position, which isfollowed by the alternate amino acid that is present at that position.For example, Y407T means that the tyrosine normally present at EUposition 407 is replaced by a threonine. Alternatively or in addition tosuch alterations, substitutions creating new disulfide bridges canfacilitate heterodimer formation. See, e.g., US Patent ApplicationPublication 2003/0078385, the portions of which describe such mutationsare incorporated herein by reference. Such alterations in an IgG1 Fcregion include, for example, the following substitutions: Y349C in oneFc polypeptide chain and S354C in the other; Y349C in one Fc polypeptidechain and E356C in the other; Y349C in one Fc polypeptide chain andE357C in the other; L351C in one Fc polypeptide chain and S354C in theother; T394C in one Fc polypeptide chain and E397C in the other; orD399C in one Fc polypeptide chain and K392C in the other. Similarly,substitutions changing the charge of a one or more residue, for example,in the C_(H)3-C_(H)3 interface, can enhance heterodimer formation asexplained in WO 2009/089004, the portions of which describe suchsubstitutions are incorporated herein by reference. Such substitutionsare referred to herein as “charge pair substitutions,” and an Fc regioncontaining one pair of charge pair substitutions contains onesubstitution in the A chain and a different substitution in the B chain.General examples of charge pair substitutions include the following: 1)K409D or K409E in one chain plus D399K or D399R in the other; 2) K392Dor K392E in one chain plus D399K or D399R in the other; 3) K439D orK439E in one chain plus E356K or E356R in the other; and 4) K370D orK370E in one chain plus E357K or E357R in the other. In addition, thesubstitutions R355D, R355E, K360D, or K360R in both chains can stabilizeheterodimers when used with other heterodimerizing alterations. Specificcharge pair substitutions can be used either alone or with other chargepair substitutions. Specific examples of single pairs of charge pairsubstitutions and combinations thereof include the following: 1) K409Ein one chain plus D399K in the other; 2) K409E in one chain plus D399Rin the other; 3) K409D in one chain plus D399K in the other; 4) K409D inone chain plus D399R in the other; 5) K392E in one chain plus D399R inthe other; 6) K392E in one chain plus D399K in the other; 7) K392D inone chain plus D399R in the other; 8) K392D in one chain plus D399K inthe other; 9) K409D and K360D in one chain plus D399K and E356K in theother; 10) K409D and K370D in one chain plus D399K and E357K in theother; 11) K409D and K392D in one chain plus D399K, E356K, and E357K inthe other; 12) K409D and K392D on one chain and D399K on the other; 13)K409D and K392D on one chain plus D399K and E356K on the other; 14)K409D and K392D on one chain plus D399K and D357K on the other; 15)K409D and K370D on one chain plus D399K and D357K on the other; 16)D399K on one chain plus K409D and K360D on the other; and 17) K409D andK439D on one chain plus D399K and E356K on the other. Any of the theseheterodimerizing alterations can be used in the Fc regions of theheterodimeric bispecific antibodies described herein.

An “alteration that inhibits FcγR binding,” as meant herein, is one ormore insertions, deletions, or substitutions within an Fc polypeptidechain that inhibits the binding of FcγRIIA, FcγRIIB, and/or FcγRIIIA asmeasured, for example, by an ALPHALISA®-based competition binding assay(PerkinElmer, Waltham, Mass.). Such alterations can be included in an Fcpolypeptide chain that is part of a Bi-Fc. More specifically,alterations that inhibit Fc gamma receptor (FcγR) binding include L234A,L235A, or any alteration that inhibits glycosylation at N297, includingany substitution at N297. In addition, along with alterations thatinhibit glycosylation at N297, additional alterations that stabilize adimeric Fc region by creating additional disulfide bridges are alsocontemplated. Further examples of alterations that inhibit FcγR bindinginclude a D265A alteration in one Fc polypeptide chain and an A327Qalteration in the other Fc polypeptide chain. Some such mutations aredescribed in, e.g., Xu et al. (2000), Cellular Immunol. 200: 16-26, theportions of which describe such mutations and how their activity isassessed are incorporated herein by reference.

An “alteration that enhances ADCC,” as meant herein is one or moreinsertions, deletions, or substitutions within an Fc polypeptide chainthat enhances antibody dependent cell-mediated cytotoxicity (ADCC). Suchalterations can be included in an Fc polypeptide chain that is part of aBi-Fc. Many such alterations are described in International PatentApplication Publication WO 2012/125850. Portions of this applicationthat describe such alterations are incorporated herein by reference.Such alterations can be included in an Fc polypeptide chain that is partof a heterodimeric bispecific antibody as described herein. ADCC assayscan be performed as follows. Cell lines that express high and loweramounts of a cancer cell antigen on the cell surface can be used astarget cells. These target cells can be labeled with carboxyfluoresceinsuccinimidyl ester (CFSE) and then washed once with phosphate bufferedsaline (PBS) before being deposited into 96-well microtiter plates withV-shaped wells. Purified immune effector cells, for example T cells, NKcells, macrophages, neutrophils can be added to each well. Amonospecific antibody that binds to the cancer antigen and contains thealteration(s) being tested and an isotype-matched control antibody canbe diluted in a 1:3 series and added to the wells. The cells can beincubated at 37° C. with 5% CO₂ for 3.5 hrs. The cells can be spun downand re-suspended in 1×FACS buffer (1× phosphate buffered saline (PBS)containing 0.5% fetal bovine serum (FBS)) with the dye TO-PRO®-3 iodide(Molecular Probes, Inc. Corporation, Oregon, USA), which stains deadcells, before analysis by fluorescence activated cell sorting (FACS).The percentage of cell killing can be calculated using the followformula:

(percent tumor cell lysis with bispecific−percent tumor cell lysiswithout bispecific)/(percent total cell lysis−percent tumor cell lysiswithout bispecific)

Total cell lysis is determined by lysing samples containing effectorcells and labeled target cells without a bispecific molecule with cold80% methanol. Exemplary alterations that enhance ADCC include thefollowing alterations in the A and B chains of an Fc region: (a) the Achain comprises Q311M and K334V substitutions and the B chain comprisesL234Y, E294L, and Y296W substitutions or vice versa; (b) the A chaincomprises E233L, Q311M, and K334V substitutions and the B chaincomprises L234Y, E294L, and Y296W substitutions or vice versa; (c) the Achain comprises L234I, Q311M, and K334V substitutions and the B chaincomprises L234Y, E294L, and Y296W substitutions or vice versa; (d) the Achain comprises S298T and K334V substitutions and the B chain comprisesL234Y, K290Y, and Y296W substitutions or vice versa; (e) the A chaincomprises A330M and K334V substitutions and the B chain comprises L234Y,K290Y, and Y296W substitutions or vice versa; (f) the A chain comprisesA330F and K334V substitutions and the B chain comprises L234Y, K290Y,and Y296W substitutions or vice versa; (g) the A chain comprises Q311M,A330M, and K334V substitutions and the B chain comprises L234Y, E294L,and Y296W substitutions or vice versa; (h) the A chain comprises Q311M,A330F, and K334V substitutions and the B chain comprises L234Y, E294L,and Y296W substitutions or vice versa; (i) the A chain comprises S298T,A330M, and K334V substitutions and the B chain comprises L234Y, K290Y,and Y296W substitutions or vice versa; (j) the A chain comprises S298T,A330F, and K334V substitutions and the B chain comprises L234Y, K290Y,and Y296W substitutions or vice versa; (k) the A chain comprises S239D,A330M, and K334V substitutions and the B chain comprises L234Y, K290Y,and Y296W substitutions or vice versa; (l) the A chain comprises S239D,S298T, and K334V substitutions and the B chain comprises L234Y, K290Y,and Y296W substitutions or vice versa; (m) the A chain comprises a K334Vsubstitution and the B chain comprises Y296W and S298C substitutions orvice versa; (n) the A chain comprises a K334V substitution and the Bchain comprises L234Y, Y296W, and S298C substitutions or vice versa; (o)the A chain comprises L235S, S239D, and K334V substitutions and the Bchain comprises L234Y, K290Y, and Y296W, substitutions or vice versa;(p) the A chain comprises L235S, S239D, and K334V substitutions and theB chain comprises L234Y, Y296W, and S298C substitutions or vice versa;(q) the A chain comprises Q311M and K334V substitutions and the B chaincomprises L234Y, F243V, and Y296W substitutions or vice versa; (r) the Achain comprises Q311M and K334V substitutions and the B chain comprisesL234Y, K296W, and S298C substitutions or vice versa; (s) the A chaincomprises S239D and K334V substitutions and the B chain comprises L234Y,K290Y, and Y296W substitutions or vice versa; (t) the A chain comprisesS239D and K334V substitutions and the B chain comprises L234Y, Y296W,and S298C substitutions or vice versa; (u) the A chain comprises F243Vand K334V substitutions and the B chain comprises L234Y, K290Y, andY296W, substitutions or vice versa; (v) the A chain comprises F243V andK334V substitutions and the B chain comprises L234Y, Y296W, and S298Csubstitutions or vice versa; (w) the A chain comprises E294L and K334Vsubstitutions and the B chain comprises L234Y, K290Y, and Y296Wsubstitutions or vice versa; (x) the A chain comprises E294L and K334Vsubstitutions and the B chain comprises L234Y, Y296W, and S298Csubstitutions or vice versa; (y) the A chain comprises A330M and K334Vsubstitutions and the B chain comprises L234Y and Y296W substitutions orvice versa; or (z) the A chain comprises A330M and K334V substitutionsand the B chain comprises K290Y and Y296W substitutions or vice versa.

An “IgG antibody,” as meant herein, is an antibody consistingessentially of two immunoglobulin IgG heavy chains and twoimmunoglobulin light chains, which can be kappa or lambda light chains.More specifically, the heavy chains contain a VH region, a CH1 region, ahinge region, a CH2 region, and a CH3 region in that order, while thelight chains contain a VL region followed by a CL region. Numeroussequences of such immunoglobulin regions are known in the art. See,e.g., Kabat et al. in SEQUENCES OF IMMUNOLOGICAL INTEREST, Public HealthService N.I.H., Bethesda, Md., 1991. Sequences of regions from IgGantibodies disclosed in Kabat et al. are incorporated herein byreference. Close variants of a known and/or naturally-occurring IgGantibody comprising no more than 10 amino acid substitutions,insertions, and/or deletions of a single amino acid per 100 amino acidsrelative to a known or naturally occurring sequence of an immunoglobulinIgG heavy and/or light chain are encompassed within what is meant by anIgG antibody.

An “immune effector cell,” as meant herein, is a cell that is involvedin the mediation of a cytolytic immune response, including, for example,T cells, NK cells, macrophages, or neutrophils. The heterodimericbispecific antibodies described herein bind to an antigen that is partof a protein expressed on the surface of an immune effector cell. Suchproteins are referred to herein as “effector cell proteins.”

An “immunoglobulin heavy chain,” as meant herein, consists essentiallyof a VH region, a CH1 region, a hinge region, a CH2 region, a CH3 regionin that order, and, optionally, a region downstream of the CH3 region insome isotypes. Close variants of an immunoglobulin heavy chaincontaining no more than 10 amino acid substitutions, insertions, and/ordeletions of a single amino acid per 100 amino acids relative to a knownor naturally occurring immunoglobulin heavy chain amino acid sequenceare encompassed within what is meant by an immunoglobulin heavy chain.

A “immunoglobulin light chain,” as meant herein, consists essentially ofa light chain variable region (VL) and a light chain constant domain(CL). Close variants of an immunoglobulin light chain containing no morethan 10 amino acid substitutions, insertions, and/or deletions of asingle amino acid per 100 amino acids relative to a known or naturallyoccurring immunoglobulin light chain amino acid sequence are encompassedwithin what is meant by an immunoglobulin light chain.

An “immunoglobulin variable region,” as meant herein, is a VH region, aVL region, or a variant thereof. Close variants of an immunoglobulinvariable region containing no more than 10 amino acid substitutions,insertions, and/or deletions of a single amino acid per 100 amino acidsrelative to a known or naturally occurring immunoglobulin variableregion amino acid sequence are encompassed within what is meant by animmunoglobulin variable region. Many examples of VH and VL regions areknown in the art, such as, for example, those disclosed by Kabat et alin SEQUENCES OF IMMUNOLOGICAL INTEREST, Public Health Service N.I.H.,Bethesda, Md., 1991. Based on the extensive sequence commonalities inthe less variable portions of the VH and VL regions, the position withina sequence of more variable regions, and the predicted tertiarystructure, one of skill in the art can recognize an immunoglobulinvariable region by its sequence. See, e.g., Honegger and Plückthun(2001), J. Mol. Biol. 309: 657-670.

An immunoglobulin variable region contains three hypervariable regions,known as complementarity determining region 1 (CDR1), complementaritydetermining region 2 (CDR2), and complementarity determining region 3(CDR3). These regions form the antigen binding site of an antibody. TheCDR5 are embedded within the less variable framework regions (FR1-FR4).The order of these subregions within an immunoglobulin variable regionis as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Numerous sequences ofimmunoglobulin variable regions are known in the art. See, e.g., Kabatet al, SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, Public HealthService N.I.H., Bethesda, Md., 1991.

CDR5 can be located in a VH region sequence in the following way. CDR1starts at approximately residue 31 of the mature VH region and isusually about 5-7 amino acids long, and it is almost always preceded bya Cys-Xxx-Xxx-Xxx-Xxx-Xxx-Xxx-Xxx-Xxx (SEQ ID NO:1) (where “Xxx” is anyamino acid). The residue following the heavy chain CDR1 is almost alwaysa tryptophan, often a Trp-Val, a Trp-Ile, or a Trp-Ala. Fourteen aminoacids are almost always between the last residue in CDR1 and the firstin CDR2, and CDR2 typically contains 16 to 19 amino acids. CDR2 may beimmediately preceded by Leu-Glu-Trp-11e-Gly (SEQ ID NO:2) and may beimmediately followed by Lys/Arg-Leu/Ile/Val/Phe/Thr/Ala-Thr/Ser/Ile/Ala.Other amino acids may precede or follow CDR2. Thirty two amino acids arealmost always between the last residue in CDR2 and the first in CDR3,and CDR3 can be from about 3 to 25 residues long. A Cys-Xxx-Xxx almostalways immediately precedes CDR3, and a Trp-Gly-Xxx-Gly (SEQ ID NO:3)almost always follows CDR3.

Light chain CDR5 can be located in a VL region in the following way.CDR1 starts at approximately residue 24 of the mature antibody and isusually about 10 to 17 residues long. It is almost always preceded by aCys. There are almost always 15 amino acids between the last residue ofCDR1 and the first residue of CDR2, and CDR2 is almost always 7 residueslong. CDR2 is typically preceded by Ile-Tyr, Val-Tyr, Ile-Lys, orIle-Phe. There are almost always 32 residues between CDR2 and CDR3, andCDR3 is usually about 7 to 10 amino acids long. CDR3 is almost alwayspreceded by Cys and usually followed by Phe-Gly-Xxx-Gly (SEQ ID NO:4).

A “linker,” as meant herein, is a peptide that links two polypeptides,which can be two immunoglobulin variable regions in the context of aheterodimeric bispecific antibody. A linker can be from 2-30 amino acidsin length. In some embodiments, a linker can be 2-25, 2-20, or 3-18amino acids long. In some embodiments, a linker can be a peptide no morethan 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 amino acids long. In otherembodiments, a linker can be 5-25, 5-15, 4-11, 10-20, or 20-30 aminoacids long. In other embodiments, a linker can be about, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 amino acids long. Exemplary linkers include, forexample, the amino acid sequences TVAAP (SEQ ID NO:17), ASTKGP (SEQ IDNO:18), GGGGSGGGGS (SEQ ID NO:19), GGGGSAAA (SEQ ID NO:20),GGGGSGGGGSGGGGS (SEQ ID NO:21), and AAA, among many others.

A Bi-Fc “mediates cytolysis of a target cell by an immune effectorcell,” as meant herein, when addition of an amount from 0.001 μM to20000 μM of the Bi-Fc to a cell cytolysis assay as described in thesection below entitled “Target Cell Cytolysis Assays” and in Example 3effectively elicits cytolysis of the target cells.

“Non-chemotherapeutic anti-neoplastic agents” are chemical agents,compounds, or molecules having cytotoxic or cytostatic effects on cancercells other than chemotherapeutic agents. Non-chemotherapeuticantineoplastic agents may, however, be targeted to interact directlywith molecules that indirectly affect cell division such as cell surfacereceptors, including receptors for hormones or growth factors. However,non-chemotherapeutic antineoplastic agents do not interfere directlywith processes that are intimately linked to cell division such as, forexample, DNA replication, RNA synthesis, protein synthesis, or mitoticspindle function, assembly, or disassembly. Examples ofnon-chemotherapeutic anti-neoplastic agents include inhibitors of Bcl2,inhibitors of farnesyltransferase, anti-estrogenic agents such astamoxifen, anti-androgenic compounds, interferon, arsenic, retinoicacid, retinoic acid derivatives, antibodies targeted to tumor-specificantigens, and inhibitors of the Bcr-Abl tyrosine kinase (e.g., the smallmolecule STI-571 marketed under the trade name GLEEVEC™ by Novartis, NewYork and New Jersey, USA and Basel, Switzerland), among many possiblenon-chemotherapeutic anti-neoplastic agents.

A “target cell” is a cell that a Bi-Fc binds to and that is involved inmediating a disease. In some cases, a target cell can be a cell that isordinarily involved in mediating an immune response, but is alsoinvolved in the mediation of a disease. For example in B cell lymphoma,a B cell, which is ordinarily involved in mediating immune response, canbe a target cell. In some embodiments, a target cell is a cancer cell, acell infected with a pathogen, or a cell involved in mediating anautoimmune or inflammatory disease, for example a fibrotic disease. TheBi-Fc can bind to the target cell via binding to an antigen on a “targetcell protein,” which is a protein that is displayed on the surface ofthe target cell, possibly a highly expressed protein.

“Tumor burden” refers to the number of viable cancer cells, the numberof tumor sites, and/or the size of the tumor(s) in a patient sufferingfrom a cancer. A reduction in tumor burden can be observed, for example,as a reduction in the amount of a tumor-associated antigen or protein ina patient's blood or urine, a reduction in the number of tumor cells ortumor sites, and/or a reduction in the size of one or more tumors.

A “therapeutically effective amount” of a Bi-Fc or any other drug is anamount that has the effect of, for example, reducing or eliminating thetumor burden of a cancer patient or reducing or eliminating the symptomsof any disease condition that the protein is used to treat. Atherapeutically effective amount need not completely eliminate allsymptoms of the condition, but may reduce severity of one or moresymptoms or delay the onset of more serious symptoms or a more seriousdisease that can occur with some frequency following the treatedcondition.

“Treatment” of any disease mentioned herein encompasses an alleviationof at least one symptom of the disease, a reduction in the severity ofthe disease, or the delay or prevention of disease progression to moreserious symptoms that may, in some cases, accompany the disease or leadto at least one other disease. Treatment need not mean that the diseaseis totally cured. A useful therapeutic agent needs only to reduce theseverity of a disease, reduce the severity of one or more symptomsassociated with the disease or its treatment, or delay the onset of moreserious symptoms or a more serious disease that can occur with somefrequency following the treated condition.

When it is said that a named VH/VL pair of immunoglobulin variableregions can bind to a target cell or and/or an immune effector cell“when they are part of an IgG and/or scFv antibody,” it is meant that anIgG antibody that contains the named VH region in both heavy chains andthe named VL region in both light chains and/or an scFv antibodycontaining these VH and VL regions can bind to the target cell and/orthe immune effector cell. The binding assay described in Example 2 canbe used to assess binding.

Bi-Fc Molecules

In the most general sense, a Bi-Fc can bind monovalently to twodifferent antigens and comprises one polypeptide chain or two differentpolypeptide chains having different amino acid sequences. In addition,it can bind to the neonatal Fc receptor (FcRn) at slightly acidic pH(about pH 5.5-6.0) via its Fc region. This interaction with FcRn canlengthen the half life of a molecule in vivo. The first polypeptidechain (which, in some cases, is the only polypeptide chain) comprises anFc polypeptide chain and two VH regions plus two VL regions separated bylinkers. The Fc polypeptide chain can be N-terminal or C-terminalrelative to the four immunoglobulin variable regions, and it can bejoined to the variable regions via a linker. The second polypeptidechain, when present, comprises an Fc polypeptide chain. A Bi-Fc can bindto an immune effector cell and a target cell and/or can mediatecytolysis of a target cell by an immune effector cell. The generalstructure of a Bi-Fc is diagrammed in FIG. 1, which shows an embodimentwhere the Fc polypeptide chain is C-terminal (at left) and an embodimentwhere the Fc polypeptide chain in N-terminal (at right).

More particular embodiments specify the order of immunoglobulin variableregions and the length of the linkers and specify which immunoglobulinvariable regions can associate to form a binding site for an effectorcell protein or a target cell protein. Generally, the antigen-bindingportion of an antibody includes both a VH and a VL region, referred toherein as a “VH/VL pair,” although in some cases a VH or a VL region canbind to an antigen without a partner. See, e.g., US ApplicationPublication 2003/0114659.

In one group of embodiments, the four variable regions can be arrangedin the following order: VH1-linker1-VL1-linker2-VH2-linker3-VL2, whereVH1/VL1 is an antigen-binding pair and VH2/VL2 is anotherantigen-binding pair. In this group of embodiments, linker1 and linker3can be at least 15 amino acids long, and linker2 can be less than 12amino acids long. In some embodiments, the VH1/VL1 pair can bind to atarget cell protein, and the VH2/VL2 pair can bind to an effector cellprotein. In other embodiments, the VH1/VL1 pair can bind to an effectorcell protein, and the VH2/VL2 pair can bind to a target cell protein.

In another group of embodiments the four variable regions can bearranged in the following order:VL1-linker1-VH1-linker2-VL2-linker3-VH2, where VH1/VL1 is anantigen-binding pair and VH2/VL2 is an antigen-binding pair. In theseembodiments, linker2 can be less than 12 amino acids long, and linker1and linker3 can be at least 15 amino acids long. In some embodiments,the VH1/VL1 pair can bind to a target cell protein, and the VH2/VL2 paircan bind to an effector cell protein. In other embodiments, the VH1/VL1pair can bind to an effector cell protein, and the VH2/VL2 pair can bindto a target cell protein.

In another group of embodiments the four variable regions can bearranged in the following order:VH1-linker1-VL1-linker2-VL2-linker3-VH2, where VH1/VL1 is anantigen-binding pair and VH2/VL2 is an antigen-binding pair. In theseembodiments, linker2 can be less than 12 amino acids long, and linker1and linker3 can be at least 15 amino acids long. In some embodiments,the VH1/VL1 pair can bind to a target cell protein, and the VH2/VL2 paircan bind to an effector cell protein. In other embodiments, the VH1/VL1pair can bind to an effector cell protein, and the VH2/VL2 pair can bindto a target cell protein.

In further group of embodiments the four variable regions can bearranged in the following order:VL1-linker1-VH1-linker2-VH2-linker3-VL2, where VH1/VL1 is anantigen-binding pair and VH2/VL2 is an antigen-binding pair. In theseembodiments, linker2 can be less than 12 amino acids long, and linker1and linker3 can be at least 15 amino acids long. In some embodiments,the VH1/VL1 pair can bind to a target cell protein, and the VH2/VL2 paircan bind to an effector cell protein. In other embodiments, the VH1/VL1pair can bind to an effector cell protein, and the VH2/VL2 pair can bindto a target cell protein.

A Bi-Fc can comprise an Fc polypeptide chain of an antibody. The Fcpolypeptide chain can be of mammalian (for example, human, mouse, rat,rabbit, dromedary, or new or old world monkey), avian, or shark origin.For example, the Fc polypeptide chain can be a human IgG1, IgG2, IgG3,or IgG4 Fc polypeptide chain. In addition, as explained above, an Fcpolypeptide chain can comprise a limited number of alterations. Moreparticularly, an Fc polypeptide chain can contain no more than 10insertions, deletions, and/or substitutions of a single amino acid per100 amino acids relative to a known or naturally-occurring sequence. Insome embodiments, the two Fc polypeptide chains of a heterodimeric Bi-Fccontain heterodimerizing alterations, which can be, for example, chargepair substitutions. For example, the first polypeptide chain of theBi-Fc can comprise the substitutions K409D or K409E and K392D or K392Eand the second polypeptide chain of the Bi-Fc can comprise D399K orD399R and D356K or D356R. Alternatively, the first polypeptide chain ofthe Bi-Fc can comprise D399K or D399R and D356K or D356R, and the secondpolypeptide chain of the Bi-Fc can comprise K409D or K409E and K392D orK392E. An Fc polypeptide chain can also comprise one or more “Fcalterations unfavorable to homodimer formation” and/or one or more “Fcalterations that extend half life,” as meant herein.

In monomeric embodiments of the Bi-Fc, the Bi-Fc can comprise one ormore “Fc alterations that are unfavorable to homodimer formation,” asdefined above.

Other kinds of alteration can also be part of an Fc polypeptide chainthat is part of a Bi-Fc. In one aspect, an Fc region included in a Bi-Fccan comprise one or more “alterations that inhibit the binding of an Fcgamma receptor (FcγR)” to the Fc region as defined above. In anotheraspect, an Fc region included in a Bi-Fc can comprise one or more “Fcalterations that extends half life,” as defined above. In still anotheraspect, one or more “alterations that enhance ADCC” can be included inan Fc region that is part of a Bi-Fc.

In some embodiments the amino acid sequences of the Fc polypeptides canbe mammalian, for example a human, amino acid sequences or variantsthereof that comprise not more than 10 deletions, insertions, orsubstitutions of a single amino acid per 100 amino acids of sequencerelative to a human amino acid sequence. The isotype of the Fcpolypeptide can be IgA, IgD, IgE, IgM, or IgG, such as IgG1, IgG2, IgG3,or IgG4. Table 2 below shows an alignment of the amino acid sequences ofhuman IgG1, IgG2, IgG3, and IgG4 Fc polypeptide chain sequences.

TABLE 2 Amino acid sequences of human IgG Fc regions IgG1             ----------------------------------------------- IgG2             ----------------------------------------------- IgG3             ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP IgG4             -----------------------------------------------        225       235       245       255       265       275         *         *         *         *         *         *  IgG1EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF IgG2ERKCCVE---CPPCPAPPVA-GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQF IgG3EPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQF IgG4ESKYG---PPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQF        285       295       305       315       325       335         *         *         *         *         *         * IgG1NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT IgG2NWYVDGMEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKT IgG3KWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT IgG4NWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT        345       355       365       375       385       395         *         *         *         *         *         * IgG1ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP IgG2ISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP IgG3ISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTP IgG4ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP        405       415       425       435       445         *         *         *         *         * IgG1PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 25)IgG2PMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 26)IgG3PMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK (SEQ ID NO: 27)IgG4PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 28)The numbering shown in Table 2 is according the EU system of numbering,which is based on the sequential numbering of the constant region of ahuman IgG1 antibody. Edelman et al. (1969), Proc. Natl. Acad. Sci. 63:78-85. Thus, it does not accommodate the additional length of the IgG3hinge well. It is nonetheless used herein to designate positions in anFc region because it is still commonly used in the art to refer topositions in Fc regions. The hinge regions of the IgG1, IgG2, and IgG4Fc polypeptides extend from about position 216 to about 230. It is clearfrom the alignment that the IgG2 and IgG4 hinge regions are each threeamino acids shorter than the IgG1 hinge. The IgG3 hinge is much longer,extending for an additional 47 amino acids upstream. The CH2 regionextends from about position 231 to 340, and the CH3 region extends fromabout position 341 to 447.

Naturally occurring amino acid sequences of Fc polypeptides can bevaried slightly. Such variations can include no more than 10 insertions,deletions, and/or substitutions of one amino acid per 100 amino acids ofsequence in a known or naturally-occurring amino acid sequence of an Fcpolypeptide. If there are substitutions, they can be conservative aminoacid substitutions, as defined above. The Fc polypeptides on the firstand second polypeptide chains of a Bi-Fc can differ in amino acidsequence. In some embodiments, they can include one or more“heterodimerizing alterations,” “alterations that enhance ADCC,”“alterations that inhibit FcγR binding,” “Fc alterations that areunfavorable to homodimer formation,” and/or “Fc alterations that extendhalf life,” as defined above.

A Bi-Fc can bind to an immune effector cell through an antigen that ispart of an effector cell protein and can bind to a target cell throughan antigen that is part of a target cell protein. A number of possibleeffector cell proteins are described in detail below. Similarly, anumber of possible target cell proteins is also described below. A Bi-Fccan bind to any combination of an effector cell protein and a targetcell protein.

Exemplary amino acid sequences of Bi-Fc's include the following pairs ofamino acid sequences: SEQ ID NOs:10 and 12 and SEQ ID NOs:15 and 12.

Nucleic Acids Encoding Bi-Fc Molecules

Provided are nucleic acids encoding Bi-Fc's. Numerous nucleic acidsequences encoding immunoglobulin regions including VH, VL, hinge, CH1,CH2, CH3, and CH4 regions are known in the art. See, e.g., Kabat et al.in SEQUENCES OF IMMUNOLOGICAL INTEREST, Public Health Service N.I.H.,Bethesda, Md., 1991. Using the guidance provided herein, one of skill inthe art could combine such nucleic acid sequences and/or other nucleicacid sequences known in the art to create nucleic acid sequencesencoding Bi-Fc's. Exemplary pairs of nucleic acids encoding Bi-Fc'sinclude SEQ ID NOs:11 and 13 and SEQ ID NOs:16 and 13.

In addition, nucleic acid sequences encoding Bi-Fc's can be determinedby one of skill in the art based on the amino acid sequences providedherein and elsewhere and knowledge in the art. Besides more traditionalmethods of producing cloned DNA segments encoding a particular aminoacid sequence, companies such as DNA 2.0 (Menlo Park, Calif., USA) andBlueHeron (Bothell, Wash., USA), among others, now routinely producechemically synthesized, gene-sized DNAs of any desired sequence toorder, thus streamlining the process of producing such DNAs.

Methods of Making Bi-Fc Molecules

Bi-Fc's can be made using methods well known in the art. For example,nucleic acids encoding the one or two polypeptide chains of a Bi-Fc canbe introduced into a cultured host cell by a variety of known methods,such as, for example, transformation, transfection, electroporation,bombardment with nucleic acid-coated microprojectiles, etc. In someembodiments the nucleic acids encoding a Bi-Fc can be inserted into avector appropriate for expression in the host cells before beingintroduced into the host cells. Typically such vectors can containsequence elements enabling expression of the inserted nucleic acids atthe RNA and protein levels. Such vectors are well known in the art, andmany are commercially available. The host cells containing the nucleicacids can be cultured under conditions so as to enable the cells toexpress the nucleic acids, and the resulting Bi-Fc's can be collectedfrom the cell mass or the culture medium. Alternatively, a Bi-Fc can beproduced in vivo, for example in plant leaves (see, e.g., Scheller etal. (2001), Nature Biotechnol. 19: 573-577 and references citedtherein), bird eggs (see, e.g., Zhu et al. (2005), Nature Biotechnol.23: 1159-1169 and references cited therein), or mammalian milk (see,e.g., Laible et al. (2012), Reprod. Fertil. Dev. 25(1): 315).

A variety of cultured host cells can be used including, for example,bacterial cells such as Escherichia coli or Bacillus stearothermophilus,fungal cells such as Saccharomyces cerevisiae or Pichia pastoris, insectcells such as lepidopteran insect cells including Spodoptera frugiperdacells, or mammalian cells such as Chinese hamster ovary (CHO) cells,baby hamster kidney (BHK) cells, monkey kidney cells, HeLa cells, humanhepatocellular carcinoma cells, or 293 cells, among many others.

Immune Effector Cells and Effector Cell Proteins

A Bi-Fc can bind to a molecule expressed on the surface of an immuneeffector cell (called “effector cell protein” herein) and to anothermolecule expressed on the surface of a target cell (called a “targetcell protein” herein). The immune effector cell can be a T cell, an NKcell, a macrophage, or a neutrophil. In some embodiments the effectorcell protein is a protein included in the T cell receptor (TCR)-CD3complex. The TCR-CD3 complex is a heteromultimer comprising aheterodimer comprising TCRα and TCRβ or TCRγ and TCRδ plus various CD3chains from among the CD3 zeta (CD3ζ) chain, CD3 epsilon (CD3ε) chain,CD3 gamma (CD3γ) chain, and CD3 delta (CD3δ) chain. In some embodimentsthe effector cell protein can be the human CD3 epsilon (CD3ε) chain (themature amino acid sequence of which is disclosed in SEQ ID NO:22), whichcan be part of a multimeric protein. Alternatively, the effector cellprotein can be human and/or cynomolgus monkey TCRα, TCRβ, TCRδ, TCRγ,CD3 beta (CD3β) chain, CD3 gamma (CD3γ) chain, CD3 delta (CD3δ) chain,or CD3 zeta (CD3ζ) chain.

Moreover, in some embodiments, a Bi-Fc can also bind to a CD3ε chainfrom a non-human species, such as mouse, rat, rabbit, new world monkey,and/or old world monkey species. Such species include, withoutlimitation, the following mammalian species: Mus musculus, Rattusrattus, Rattus norvegicus, the cynomolgus monkey, Macaca fascicularis,the hamadryas baboon, Papio hamadryas, the Guinea baboon, Papio papio,the olive baboon, Papio anubis, the yellow baboon, Papio cynocephalus,the Chacma baboon, Papio ursinus, Callithrix jacchus, Saguinus Oedipus;and Saimiri sciureus. The mature amino acid sequence of the CD3ε chainof cynomolgus monkey is provided in SEQ ID NO:23. As is known in the artof development of protein therapeutics, having a therapeutic that canhave comparable activity in humans and species commonly used forpreclinical testing, such as mice and monkeys, can simplify and speeddrug development. In the long and expensive process of bringing a drugto market, such advantages can be critical.

In more particular embodiments, the heterodimeric bispecific antibodycan bind to an epitope within the first 27 amino acids of the CD3εchain, which may be a human CD3ε chain or a CD3ε chain from differentspecies, particularly one of the mammalian species listed above. Theepitope can contain the amino acid sequence Gln-Asp-Gly-Asn-Glu (SEQ IDNO:24). The advantages of an antibody that binds such an epitope areexplained in detail in U.S. Patent Application Publication 2010/183615,the relevant portions of which are incorporated herein by reference. Theepitope to which an antibody binds can be determined by alaninescanning, which is described in, e.g., U.S. Patent ApplicationPublication 2010/183615, the relevant portions of which are incorporatedherein by reference.

Where a T cell is the immune effector cell, effector cell proteins towhich a Bi-Fc can bind include, without limitation, the CD3ε chain, theCD3γ, the CD3δ chain, the CD3ζ chain, TCRα, TCRβ, TCRγ, and TCRδ. Wherean NK cell or a cytotoxic T cell is an immune effector cell, NKG2D,CD352, NKp46, or CD16a can, for example, be an effector cell protein.Where a CD8⁺ T cell is an immune effector cell, 4-1BB or NKG2D, forexample, can be an effector cell protein. Alternatively, a Bi-Fc couldbind to other effector cell proteins expressed on T cells, NK cells,macrophages, or neutrophils.

Target Cells and Target Cell Proteins Expressed on Target Cells

As explained above, a Bi-Fc can bind to an effector cell protein and atarget cell protein. The target cell protein can, for example, beexpressed on the surface of a cancer cell, a cell infected with apathogen, or a cell that mediates a disease, for example aninflammatory, autoimmune, and/or fibrotic condition. In someembodiments, the target cell protein can be highly expressed on thetarget cell, although high levels of expression are not necessarilyrequired.

Where the target cell is a cancer cell, a heterodimeric bispecificantibody as described herein can bind to a cancer cell antigen asdescribed above. A cancer cell antigen can be a human protein or aprotein from another species. For example, a heterodimeric bispecificantibody may bind to a target cell protein from a mouse, rat, rabbit,new world monkey, and/or old world monkey species, among many others.Such species include, without limitation, the following species: Musmusculus, Rattus rattus, Rattus norvegicus, cynomolgus monkey, Macacafascicularis, the hamadryas baboon, Papio hamadryas, the Guinea baboon,Papio papio, the olive baboon, Papio anubis, the yellow baboon, Papiocynocephalus, the Chacma baboon, Papio ursinus, Callithrix jacchus,Saguinus oedipus, and Saimiri sciureus.

In some examples, the target cell protein can be a protein selectivelyexpressed on an infected cell. For example, in the case of an HBV or HCVinfection, the target cell protein can be an envelope protein of HBV orHCV that is expressed on the surface of an infected cell. In otherembodiments, the target cell protein can be gp120 encoded by humanimmunodeficiency virus (HIV) on HIV-infected cells.

In other aspects, a target cell can be a cell that mediates anautoimmune or inflammatory disease. For example, human eosinophils inasthma can be target cells, in which case, EGF-like module containingmucin-like hormone receptor (EMR1), for example, can be a target cellprotein. Alternatively, excess human B cells in a systemic lupuserythematosus patient can be target cells, in which case CD19 or CD20,for example, can be a target cell protein. In other autoimmuneconditions, excess human Th2 T cells can be target cells, in which caseCCR4 can, for example, be a target cell protein. Similarly, a targetcell can be a fibrotic cell that mediates a disease such asatherosclerosis, chronic obstructive pulmonary disease (COPD),cirrhosis, scleroderma, kidney transplant fibrosis, kidney allograftnephropathy, or a pulmonary fibrosis, including idiopathic pulmonaryfibrosis and/or idiotypic pulmonary hypertension. For such fibroticconditions, fibroblast activation protein alpha (FAP alpha) can, forexample, be a target cell protein.

Target Cell Cytolysis Assays

In the Examples below, an assay for determining whether a heterodimericbispecific antibody as described herein can induce cytolysis of a targetcell by an immune effector cell in vitro is described. In this assay,the immune effector cell is a T cell. The following very similar assaycan be used where the immune effector cells are NK cells.

A target cell line expressing the target cell protein of interest can belabeled with 2 μM carboxyfluorescein succinimidyl ester (CFSE) for 15minutes at 37° C. and then washed. An appropriate number of labeledtarget cells can then be incubated in one or more 96 well flat bottomculture plates for 40 minutes at 4° C., with or without a bispecificprotein, a control protein, or no added protein at varyingconcentrations. NK cells isolated from healthy human donors can beisolated using the Miltenyi NK Cell Isolation Kit II (Miltenyi Biotec,Auburn, Calif.) and then added to the target cells at an Effector:Targetratio of 10:1. The NK cells, which are the immune effector cells in thisassay, can be used immediately post-isolation or after overnight cultureat 37° C. Plates containing tumor target cells, bispecific proteins, andimmune effector cells can be cultured for 18-24 hours at 37° C. with 5%CO2. Appropriate control wells can also be set up. After the 18-24 hourassay period, all cells can be removed from the wells. A volume of a7-AAD solution equal to the volume of the content of the wells can beadded to each sample. Samples can then assayed to determine thepercentage of live versus dead target cells via flow cytometry asdescribed in the Examples below.

Therapeutic Methods and Compositions

Bi-Fc's can be used to treat a wide variety of conditions including, forexample, various forms of cancer, infections, autoimmune or inflammatoryconditions, and/or fibrotic conditions.

Provided herein are pharmaceutical compositions comprising Bi-Fc's. Suchpharmaceutical compositions comprise a therapeutically effective amountof a Bi-Fc plus one or more additional components such as aphysiologically acceptable carrier, excipient, or diluent. Suchadditional components can include buffers, carbohydrates, polyols, aminoacids, chelating agents, stabilizers, and/or preservatives, among manypossibilities.

In some embodiments, a Bi-Fc can be used to treat cell proliferativediseases, including cancer, which involve the unregulated and/orinappropriate proliferation of cells, sometimes accompanied bydestruction of adjacent tissue and growth of new blood vessels, whichcan allow invasion of cancer cells into new areas, i.e. metastasis.Included within conditions treatable with a Bi-Fc are non-malignantconditions that involve inappropriate cell growth, including colorectalpolyps, cerebral ischemia, gross cystic disease, polycystic kidneydisease, benign prostatic hyperplasia, and endometriosis. A Bi-Fc can beused to treat a hematologic or solid tumor malignancy. Morespecifically, cell proliferative diseases that can be treated using aBi-Fc are, for example, cancers including mesotheliomas, squamous cellcarcinomas, myelomas, osteosarcomas, glioblastomas, gliomas, carcinomas,adenocarcinomas, melanomas, sarcomas, acute and chronic leukemias,lymphomas, and meningiomas, Hodgkin's disease, Sézary syndrome, multiplemyeloma, and lung, non-small cell lung, small cell lung, laryngeal,breast, head and neck, bladder, ovarian, skin, prostate, cervical,vaginal, gastric, renal cell, kidney, pancreatic, colorectal,endometrial, and esophageal, hepatobiliary, bone, skin, and hematologiccancers, as well as cancers of the nasal cavity and paranasal sinuses,the nasopharynx, the oral cavity, the oropharynx, the larynx, thehypolarynx, the salivary glands, the mediastinum, the stomach, the smallintestine, the colon, the rectum and anal region, the ureter, theurethra, the penis, the testis, the vulva, the endocrine system, thecentral nervous system, and plasma cells.

Among the texts providing guidance for cancer therapy is Cancer,Principles and Practice of Oncology, 4th Edition, DeVita et al., Eds. J.B. Lippincott Co., Philadelphia, Pa. (1993). An appropriate therapeuticapproach is chosen according to the particular type of cancer, and otherfactors such as the general condition of the patient, as is recognizedin the pertinent field. A Bi-Fc can be added to a therapy regimen usingother anti-neoplastic agents in treating a cancer patient.

In some embodiments, a Bi-Fc can be administered concurrently with,before, or after a variety of drugs and treatments widely employed incancer treatment such as, for example, chemotherapeutic agents,non-chemotherapeutic, anti-neoplastic agents, and/or radiation. Forexample, chemotherapy and/or radiation can occur before, during, and/orafter any of the treatments described herein. Examples ofchemotherapeutic agents are discussed above and include, but are notlimited to, cisplatin, taxol, etoposide, mitoxantrone (Novantrone®),actinomycin D, cycloheximide, camptothecin (or water soluble derivativesthereof), methotrexate, mitomycin (e.g., mitomycin C), dacarbazine(DTIC), anti-neoplastic antibiotics such as adriamycin (doxorubicin) anddaunomycin, and all the chemotherapeutic agents mentioned above.

A Bi-Fc can also be used to treat infectious disease, for example achronic hepatitis B virus (HBV) infection, a hepatitis C virus (HCV)infection, a human immunodeficiency virus (HIV) infection, anEpstein-Barr virus (EBV) infection, or a cytomegalovirus (CMV)infection, among many others.

A Bi-Fc can find further use in other kinds of conditions where it isbeneficial to deplete certain cell types. For example, depletion ofhuman eosinophils in asthma, excess human B cells in systemic lupuserythematosus, excess human Th2 T cells in autoimmune conditions, orpathogen-infected cells in infectious diseases can be beneficial. In afibrotic condition, it can be useful to deplete cells forming fibrotictissue.

Therapeutically effective doses of a Bi-Fc can be administered. Theamount of Bi-Fc that constitutes a therapeutically dose may vary withthe indication treated, the weight of the patient, the calculated skinsurface area of the patient. Dosing of a Bi-Fc can be adjusted toachieve the desired effects. In many cases, repeated dosing may berequired. For example, a Bi-Fc can be dosed twice per week, once perweek, once every two, three, four, five, six, seven, eight, nine, or tenweeks, or once every two, three, four, five, or six months. The amountof a Bi-Fc administered on each day can be from about 0.0036 mg to about450 mg. Alternatively, the dose can calibrated according to theestimated skin surface of a patient, and each dose can be from about0.002 mg/m² to about 250 mg/m². In another alternative, the dose can becalibrated according to a patient's weight, and each dose can be fromabout 0.000051 mg/kg to about 6.4 mg/kg.

A Bi-Fc, or a pharmaceutical composition containing such a molecule, canbe administered by any feasible method. Protein therapeutics willordinarily be administered by a parenteral route, for example byinjection, since oral administration, in the absence of some specialformulation or circumstance, would lead to hydrolysis of the protein inthe acid environment of the stomach. Subcutaneous, intramuscular,intravenous, intraarterial, intralesional, or peritoneal bolus injectionare possible routes of administration. A Bi-Fc can also be administeredvia infusion, for example intravenous or subcutaneous infusion. Topicaladministration is also possible, especially for diseases involving theskin. Alternatively, a Bi-Fc can be administered through contact with amucus membrane, for example by intra-nasal, sublingual, vaginal, orrectal administration or administration as an inhalant. Alternatively,certain appropriate pharmaceutical compositions comprising a Bi-Fc canbe administered orally.

Having described the invention in general terms above, the followingexamples are offered by way of illustration and not limitation.

EXAMPLES Example 1 Construction of Anti-CD3E/HER2 andAnti-CD3E/FOLR1Bi-Fc Molecules and Single Chain Bispecific Molecules

Bi-Fc molecules were generated using methods essentially describedpreviously. Löffler et al. (2000), Blood 95(6): 2098-2103. In moredetail, a construct encoding an anti-HER2/CD3 Bi-Fc was made as follows.DNA fragments encoding the VH region (SEQ ID NO:5) and the VL region(SEQ ID NO:6) of an anti-HER2 IgG antibody and the VH region (SEQ IDNO:7) and VL region (SEQ ID NO:8) of anti-human CD3 IgG antibody wereamplified by PCR using forward and reverse primers and spliced togetherwith flexible linkers. The resulting DNA fragment, which encodes alinear fusion DNA encoding two scFv's joined by a linker is referred toherein as the single chain anti-HER2/CD3 (SEQ ID NO:9). This constructwas subcloned into a mammalian expression vector for antibodyproduction.

An anti-HER2/CD3 Bi-Fc (SEQ ID NO:10) was constructed by fusing DNAencoding the single chain anti-HER2/CD3 to DNA encoding one of the twochains of an engineered human IgG1 Fc region. Specifically, DNA encodingan Fc polypeptide chain containing two positively charged mutations(D356K/D399K, EU numbering) plus alterations that inhibit FcγR binding(L234A and L235A) was fused to the DNA encoding the single chainanti-HER2/CD3 at the 3′ end. The amino acid sequence of thisanti-HER/CD3 Bi-Fc and the nucleic acid sequence encoding it are shownin SEQ ID NO:10 and 11, respectively. The second polypeptide chain thatwas part of the anti-HER2/CD3 Bi-Fc was a human IgG1 Fc polypeptidechain containing two negatively charged mutations (K392D/K409D, EUnumbering) plus L234A and L235A, as shown in SEQ ID NO:12. DNA encodingthis polypeptide (SEQ ID NO:13) was amplified and inserted into anappropriate vector for expression. Using similar methods, a single chainanti-FOLR1/CD3 (SEQ ID NO: 14) and an anti-FOLR1/CD3 Bi-Fc (SEQ IDNO:15) were constructed by replacing DNA encoding the anti-HER2 scFvfragment with DNA encoding an scFv fragment derived from an anti-humanFOLR1 IgG antibody.

All single chain and Bi-Fc molecules described above were produced bytransient transfection in human HEK 293-6E cells. The culture media washarvested after 6 days. The single chain anti-HER2/CD3 andanti-FOLR1/CD3 molecules were purified by nickel HISTRAP® (GE HealthcareBio-Sciences, L.L.C., Uppsala, Sweden) column chromatography and elutedwith a 25 to 300 mM imidizole gradient. The elution pools were furtherpurified by size exchange chromatography (SEC) using a preparativeSUPERDEX® 200 (GE Healthcare Bio-Sciences, L.L.C., Uppsala, Sweden)column, concentrated to >1 mg/mL, and stored at −70° C. Anti-HER2/CD3Bi-Fc and anti-FOLR1/CD3 Bi-Fc molecules were purified using MABSELECTSURE™ (GE Healthcare Bio-Sciences, L.L.C., Uppsala, Sweden) affinitychromatography, eluting with 50 mM citrate, 1M L-Arginine, pH 3.5. Theeluate was buffer-exchanged into formulation buffer by a preparative SECwith 10 mM potassium phosphate, 161 mM L-Arginine, pH 7.6 or with asolution containing acetate and sucrose with 150 mM NaCl, 161 mML-Arginine, pH 5.2

Example 2 Testing BiTE:Fc Molecules for Binding to Target Cells andImmune Effector Cells

Binding of the anti-HER2/CD3 Bi-Fc and single chain anti-HER2/CD3 to Tcells expressing CD3 and JIMT-1 cells expressing HER2 was assessed asfollows. Human pan-T cells (purified using Pan T Cell Isolation Kit II,human, Miltenyi Biotec, Auburn, Calif.) or purified JIMT-1 cells wereincubated for 16 hrs at 4° C. in the absence or presence of 10 μg/mL ofthe anti-HER2/CD3 Bi-Fc or the single chain anti-HER2/CD3. Cell bindingof the anti-HER2/CD3 Bi-Fc was detected using an allophycocyanin(APC)-labeled anti-human Fc secondary antibody. The single chainanti-HER2/CD3, which includes a FLAG tag, was detected using a mouseanti-FLAG® antibody followed by an APC-labeled mouse Ig-specificantibody.

In the fluorescence-activated cell sorting (FACS) histograms shown inFIG. 2, the unfilled profiles represent data from cells in the absenceof one of the bispecific molecules, and the solidly filled profilesrepresent data from cells in the presence of one of the bispecificmolecules, as indicated in the description of FIG. 2. These resultsindicate that the anti-HER2/CD3 Bi-Fc, as well as the single chainanti-HER2/CD3, binds to both T cells (expressing CD3) and to JIMT-1cells expressing HER2.

Example 3 Lysis of Tumor Cell Lines in the Presence of Bi-Fc's and TCells

The anti-HER2/CD3ε and anti-FOLR1/CD3ε Bi-Fc's and single chainanti-HER2/CD3ε and anti-FOLR1/CD3ε molecules described above wereassayed to determine their activity in a T cell-dependent cell cytolysis(TDCC) assay using tumor cells expressing HER2 or FOLR1 as target cells.Briefly, pan T cells were isolated from healthy human donors using thePan T Cell Isolation Kit II, human (Miltenyi Biotec, Auburn, Calif.).The T cells were incubated with CFSE-labeled tumor target cells at aratio of 10:1 in the presence or absence of the anti-HER2/CD3ε oranti-FOLR1/CD3ε Bi-Fc's or the single chain anti-HER2/CD3ε oranti-FOR1/CD3 described in Example 1 at the varying concentrations asindicated in FIGS. 2 and 3. As a control, some samples contained T cellsand tumor target cells, but no Bi-Fc or single chain molecule.

The target cells for the anti-HER2/CD3ε Bi-Fc and single chain moleculewere either Cal-51 cells (expressing about 148,000 molecules of FOLR1per cell), T47D cells (expressing about 101,000 molecules of FOLR1 percell), or the control cell line BT474 (which did not express detectablelevels of FOLR1).

The target cells for the anti-CD3ε/HER2 Bi-Fc and single chain moleculeswere JIMT-1 cells (expressing about 181,000 molecules of HER2 per cell),T47D cells (expressing about 61,000 molecules of HER2 per cell), or thecontrol cell line SHP77 (which did not express detectable amounts ofHER2).

After 39 to 48 hours of incubation, cells were harvested, and thepercent of tumor cell lysis was monitored by uptake of7-amino-actinomycin D (7-AAD), which stains double-stranded nucleicacids. Intact cells exclude 7-AAD, whereas 7-AAD can penetrate themembranes of dead or dying cells and stain the double-stranded nucleicacids inside these cells. Percent specific lysis was calculatedaccording to the following formula:

% specific lysis=[% tumor lysis with Bi-Fc−% tumor cell lysis withoutbispecific/% of total cell lysis−% tumor cell lysis withoutbispecific]×100.

To determine percent total cell lysis, samples containing immuneeffector and labeled target cells without a Bi-Fc or single chainmolecule were lysed with cold 80% methanol.

Results for the anti-FOLR1/CD3ε Bi-Fc and single chain molecule areshown in FIG. 3. Both the anti-FOLR1/CD3ε Bi-Fc and single chainmolecule exhibited dose dependent lysis of both the Cal-51 and the T47Dtarget cells. The EC₅₀ for each of these molecules in each of these celllines is shown in Table 3 below.

TABLE 3 EC₅₀ of Bi-Fc and single chain anti-FOLR1/CD3 molecules EC₅₀(pM) Cell Line Molecule Cal-51 T47D B7474 Anti-FOLR1/CD3ε Bi-Fc 1.271.35 NA* Anti-FOLR1/CD3ε single 0.087 0.19 NA* chain *NA means thatthere was little or no cell lysis detected.These data indicate that both the anti-FOLR1/CD3ε Bi-Fc and single chainmolecule can mediate lysis of cells expressing FOLR1 in the presence ofT cells, but do not mediate lysis of cells not expressing FOLR1. TheEC₅₀'s of the s Bi-Fc are about 10 fold higher than those of the singlechain molecule, but they are still in the pM range. Thus, both the Bi-Fcand the single chain molecule are highly potent in this assay.

Results for the anti-HER2/CD3ε Bi-Fc and single chain molecule are shownin FIG. 4. Both the anti-HER2/CD3ε Bi-Fc and single chain moleculeexhibited dose dependent lysis of both the JIMT-1 and the T47D targetcells, but no lysis of the control SHP77 cell line (which does notexpress HER2). The EC₅₀ for each of these molecules in each of thesecell lines is shown in Table 4 below.

TABLE 4 EC₅₀ of Bi-Fc and single chain anti-FOLR1/CD3 molecules EC₅₀(pM) Cell Line Molecule JIMT-1 T47D SHP77 Anti-HER2/CD3ε BiFc 11.52 1.03NA* Anti-HER2/CD3ε single chain 1.12 0.12 NA* *NA means that there waslittle or no cell lysis detected.These data indicate that both the anti-HER2/CD3ε Bi-Fc and single chainmolecule can mediate lysis of cells expressing HER2 in the presence of Tcells, but do not mediate lysis of cells not expressing HER2. The EC₅₀'sof the BiFc's are about 10 fold higher than those of the single chainmolecule.

Example 4 Release of Cytokines by T Cells in the Presence of Bi-Fc andTarget Cells

The anti-HER2/CD3ε single chain and Bi-Fc and the anti-FOLR1/CD3ε singlechain and Bi-Fc described above were assayed to determine whether theycould stimulate the production of inflammatory cytokines by T cells.Briefly, twenty four hour cell culture supernatants from the TDCC assayslike those described in Example 3 were assessed for cytokineconcentrations using the Human TH1/TH2 7-Plex and Human Proinflammatory1 4-Plex ultra Sensitive Kits from Meso Scale Diagnostics, L.L.C. Assayswere performed according to the manufacturer's directions.

These results are shown in FIGS. 5 and 6. As shown in FIG. 5, PanelsA-E, the T cells secreted cytokines in the presence of theanti-FOLR1/CD3ε Bi-Fc or single chain in the presence of cellsexpressing FOLR1 (T47D, graphs on the left), but not in the presence ofcells that did not express FOLR1 (BT474, graphs on the right).Similarly, as shown in FIG. 6, Panels A-E, T cells secreted cytokines inthe presence of the anti-HER2/CD3ε Bi-Fc or single chain in the presenceof cells expressing HER2 (JIMT-1, graphs on the left), but not in thepresence of cells that did not express HER2 (SHP77, graphs on theright). Thus, the secretion of interferon gamma (IFN-γ), tumor necrosisfactor alpha (TNF-α), interleukin-10 (IL-10), interleukin-2 (IL-2), andinterleukin-13 (IL-13) by T cells in the presence of a Bi-Fc or singlechain molecule was dependent on the presence of cells expressing atarget cell protein. Hence, activation of the T cells by the Bi-Fc's andsingle chain molecules was specific in the sense that it occurred onlyin the presence of target cells expressing a target cell protein.

In addition, the Bi-Fc's had very potent activity in the assay,exhibiting EC₅₀'s in the pM range as shown in the table below.

TABLE 5 EC₅₀'s for eliciting cytokine secretion EC₅₀ (pM) JIMT-1 cellsT47D cells anti-HER2/ anti-HER2/ anti-FOLR1/ anti-FOLR1/ CD3ε CD3ε CD3εCD3ε Cytokine Bi-Fc single chain Bi-Fc single chain IFN-γ 32.9 2.1 48.67.5 TNF-α 19.5 1.8 41.2 8.8 IL-10 9.6 0.9 110.1 18.4 IL-2 22.3 1.2 67.312.9 IL-13 16.4 1.8 126.9 28.1Thus, even though the Bi-Fc is almost twice the size of the single chainmolecule, it remains a very potent activator of cytokine secretion by Tcells in the presence, but not in the absence of, target cells. Inaddition, the Bi-Fc and the single chain molecule show a very similarcytokine profile.

Example 5 Upregulation of T Cell Activation Markers in the Presence ofBi-Fc and Target Cells

The following experiment was done to determine whether a Bi-Fc couldactivate T cells in the presence of peripheral blood mononuclear cells(PBMC) and in the presence or absence of target cells. PBMC from healthydonors were purified on a FICOLL™ gradient from human leukocytespurchased from Biological Specialty Corporation of Colmar, Pa. ThesePBMC were incubated with the anti-HER2/CD3ε Bi-Fc or the single chainanti-HER2/CD3 bispecific molecule described above in the presence orabsence of JIMT-1 cells at a 10:1 ratio. After 48 hours of incubation,non-adherent cells were removed from the wells and divided into twoequal samples. All samples were stained with fluorescein isothiocynate(FITC)-conjugated anti-human CD3 antibody plus an allophycocyanin(APC)-conjugated anti-CD25 or anti-CD69 antibody. CD25 and CD69 aremarkers of activation of T cells.

Up-regulation of CD25 and CD69 (FIG. 7) activation markers by CD3⁺peripheral T cells was observed with the anti-HER2/CD3ε Bi-Fc and theanti-HER2/CD3 single chain in the presence, but not in the absence, ofHER2-expressing JIMT-1 tumor target cells. These observations suggestthat T cell activation by the Bi-Fc is dependent on the presence oftumor target cells expressing the target cell protein and thatactivation of T cells in the peripheral blood through cross-linking byFcγR's in the presence of a Bi-Fc, similar to the anti-HER2/CD3ε Bi-Fc,likely not occur. The Fc region of the anti-HER2/CD3ε Bi-Fc containsalterations that inhibit binding to FcgRs.

Example 6 Pharmacokinetic Properties of Bi-Fc's

In the following experiment, the single dose pharmacokinetic profiles ofan anti-HER2/CD3ε Bi-Fc (comprising the amino acid sequences of SEQ IDNOs:10 and 12) and an anti-HER2/CD3ε single chain (comprising the aminoacid sequence of SEQ ID NO:9) was assessed by intravenous andsubcutaneous bolus administration in male NOD.SCID mice (Harlan,Livermore, Calif.). These test molecules were injected as a bolus at 1mg/kg intravenously via the lateral tail vein in some mice orsubcutaneously under the skin over the shoulders in others. Serialbleeds of approximately 0.1 mL of whole blood were collected at eachtime point via retro-orbital sinus puncture. Upon clotting of wholeblood the samples were processed to obtain serum (˜0.040 mL per sample).Serum samples were analyzed by immunoassay using the technology Gyros AB(Warren, N.J.) to determine the serum concentrations of theanti-HER2/CD3ε single chain and Bi-Fc. Serum samples were collected at0, 0.5, 2, 8, 24, 72, 120, 168, 240, 312, 384, and 480 hours. Serumsamples were maintained at −70° C. (±10° C.) prior to analysis.Pharmacokinetic parameters were estimated from serum concentrationsusing non-compartmental analysis using Phoenix® 6.3 software (Pharsight,Sunnyvale, Calif.).

The single dose pharmacokinetic profiles of the Bi-Fc and the singlechain molecule are shown in FIG. 8. The Bi-Fc showed an extended serumhalf life (219 hours) compared to the single chain molecule, which wasrapidly eliminated and had a half life of only 5 hours. Exposure of theBi-Fc was characterized by an area under the curve (AUC) of 524hr*μg/mL, as compared to 19 hr*μg/mL for the single chain molecule. Thesubcutaneous bioavailability of the Bi-Fc was 83%, while that of thesingle chain molecule was 29%. Thus, the Bi-Fc showed favorable singledose pharmacokinetic properties as compared to the single chainmolecule.

What is claimed is:
 1. A Bi-Fc, which comprises (a) a polypeptide chaincomprising an amino acid sequence having the following formula:V1-L1-V2-L2-V3-L3-V4-L4-Fc; wherein two of V1, V2, V3, and V4 are heavychain variable (VH) regions and the other two are light chain variable(VL) regions; wherein Fc is a human IgG Fc polypeptide chain; whereinL1, L2, L3, and L4 are linkers; and wherein L4 can be present or absent;or (b) a polypeptide chain comprising an amino acid sequence having thefollowing formula: Fc-L4-V1-L1-V2-L2-V3-L3-V4; wherein two of V1, V2,V3, and V4 are heavy chain variable (VH) regions and the other two arelight chain variable (VL) regions; wherein Fc is a human IgG Fcpolypeptide chain; wherein L1, L2, L3, and L4 are linkers; and whereinL4 can be present or absent; wherein the Bi-Fc binds to a target celland an immune effector cell and/or mediates cytolysis of a target celldisplaying a target cell protein by an immune effector cell, and whereinthe Bi-Fc is a monomer.
 2. The Bi-Fc of claim 1, wherein Fc polypeptidechain of (a) or (b) comprises one or more the following alterations:K392D, K392E, K409D, K409E, D399K, D399R, E356R, E356K, D356R, D356K,Y349T, L351T, L368T, L398T, F405T, Y407T, and Y407R.
 3. The Bi-Fc ofclaim 2, wherein Fc polypeptide chain of (a) or (b) comprises K392D,K409D, and Y349T.
 4. The Bi-Fc of claim 2, wherein the Fc polypeptidechain of the polypeptide chain of (a) or (b) comprises one or morealteration that inhibits FcγR binding and/or one or more alterationsthat extend(s) half life.
 5. The Bi-Fc of claim 1, which is the Bi-Fc ofclaim 1(a).
 6. The Bi-Fc of claim 1, which is the Bi-Fc of claim 1(b).7. The Bi-Fc of claim 1, wherein the immune effector cell is a human Tcell and/or a cynomolgus monkey T cell and the effector cell protein ispart of the human and/or cynomolgus monkey T cell receptor (TCR)-CD3complex.
 8. The Bi-Fc of claim 7, wherein the effector cell protein ishuman or cynomolgus monkey CD3ε.
 9. The Bi-Fc of claim 8, wherein theamino acid sequence to which the Bi-Fc binds comprisesGln-Asp-Gly-Asn-Glu (SEQ ID NO:24) as determined by alanine scanning.10. The Bi-Fc of claim 8, wherein the Bi-Fc comprises: the amino acidsequences of the CDR1, CDR2 and CDR3 of the VH region comprising theamino acid sequence of SEQ ID NO:7 or 29; and the amino acid sequencesof the CDR1, CDR2 and CDR3 of the VL region comprising the amino acidsequence of SEQ ID NO:8 or
 31. 11. The Bi-Fc of claim 8, wherein theBi-Fc comprises: a VH region comprising the amino acid sequence of SEQID NO:7 or 29 or a variant thereof comprising no more than 10 amino acidsubstitutions, insertions, and/or deletions of a single amino acid per100 amino acids relative to SEQ ID NO:7 or 29; and a VL regioncomprising the amino acid sequence of SEQ ID NO:8 or 31 or a variantthereof comprising no more than 10 amino acid substitutions, insertions,and/or deletions of a single amino acid per 100 amino acids relative toSEQ ID NO:8 or
 31. 12. The Bi-Fc of claim 11, comprising the amino acidsequence of SEQ ID NO:7 or 29 and the amino acid of SEQ ID NO:8 and 31.13. The Bi-Fc of claim 1, wherein the target cell is a cancer cell, acell infected by a pathogen, or a cell that mediates disease.
 14. TheBi-Fc of claim 13, wherein the target cell is a cell infected by apathogen, wherein the pathogen is selected from the group consisting ofhuman immunodeficiency virus, hepatitis virus, human papilloma virus,cytomegalovirus, or a bacterium of the genus Listeria, Mycobacterium,Staphylococcus, or Streptococcus, or wherein the target cell is afibrotic cell that mediates a fibrotic disease.
 15. The Bi-Fc of claim13, wherein the target cell is a cancer cell.
 16. A Bi-Fc, whichcomprises (i) a first polypeptide comprising an amino acid sequencehaving the following formula: V1-L1-V2-L2-V3-L3-V4-L4-Fc; wherein Fc isa human IgG Fc polypeptide chain; wherein V1, V2, V3 and V4 areimmunoglobulin variable regions; wherein L1, L2, L3, and L4 are linkers;and wherein L4 can be present or absent; and (ii) a second polypeptidecomprising a human IgG Fc polypeptide chain; wherein L1 and L3 are atleast 15 amino acids long and L2 is less than 12 amino acids long,wherein either V1 is a VH region and V2 is a VL region or vice versa,wherein either V3 is a VH region and V4 is a VL region or vice versa,wherein the Bi-Fc binds to a target cell and an immune effector celland/or mediates cytolysis of a target cell displaying a target cellprotein by an immune effector cell, wherein the Fc polypeptide chain inthe first polypeptide chain comprises the charge pair substitutionsK409E or K409E and K392D or K392E, and the Fc polypeptide chain in thesecond polypeptide chain comprises the charge pair substitutions D399Kor D399R and D356K or D356R, or wherein the Fc polypeptide chain in thesecond polypeptide chain comprises the charge pair substitutions K409Eor K409E and K392D or K392E, and the Fc polypeptide chain in the firstpolypeptide chain comprises the charge pair substitutions D399K or D399Rand D356K or D356R, and wherein the Bi-Fc comprises: a VH regioncomprising the amino acid sequence of SEQ ID NO:7 or 29 or a variantthereof comprising no more than 10 amino acid substitutions, insertions,and/or deletions of a single amino acid per 100 amino acids relative toSEQ ID NO:7 or 29; and a VL region comprising the amino acid sequence ofSEQ ID NO:8 or 31 or a variant thereof comprising no more than 10 aminoacid substitutions, insertions, and/or deletions of a single amino acidper 100 amino acids relative to SEQ ID NO:8 or
 31. 17. A nucleic acidencoding the Bi-Fc of claim
 1. 18. A vector comprising the nucleic acidof claim
 17. 19. A host comprising the nucleic acid of claim 17 or thevector of claim
 18. 20. One or more nucleic acid(s) encoding the Bi-Fcof claim
 16. 21. One or more vector(s) comprising the nucleic acid(s) ofclaim
 20. 22. A host cell containing the nucleic acid(s) of claim 20 orthe vector(s) of claim
 21. 23. A method of making a Bi-Fc comprisingculturing a host cell under conditions such that a nucleic acid in thehost cell encoding the Bi-Fc is expressed, and recovering the Bi-Fc fromthe cell mass or the culture medium, wherein the Bi-Fc comprises: (a)(i) a polypeptide chain comprising an amino acid sequence having thefollowing formula: V1-L1-V2-L2-V3-L3-V4-L4-Fc; wherein two of V1, V2,V3, and V4 are heavy chain variable (VH) regions and the other two arelight chain variable (VL) regions; wherein Fc is a human IgG Fcpolypeptide chain; wherein L1, L2, L3, and L4 are linkers; and whereinL4 can be present or absent; or (ii) a polypeptide chain comprising anamino acid sequence having the following formula:Fc-L4-V1-L1-V2-L2-V3-L3-V4; wherein two of V1, V2, V3, and V4 are heavychain variable (VH) regions and the other two are light chain variable(VL) regions; wherein Fc is a human IgG Fc polypeptide chain; whereinL1, L2, L3, and L4 are linkers; and wherein L4 can be present or absent;wherein the Bi-Fc binds to a target cell and an immune effector celland/or mediates cytolysis of a target cell displaying a target cellprotein by an immune effector cell, and wherein the Bi-Fc is a monomer;or (b) (i) a first polypeptide comprising an amino acid sequence havingthe following formula: V1-L1-V2-L2-V3-L3-V4-L4-Fc; wherein Fc is a humanIgG Fc polypeptide chain; wherein V1, V2, V3 and V4 are immunoglobulinvariable regions; wherein L1, L2, L3, and L4 are linkers; and wherein L4can be present or absent; and (ii) a second polypeptide comprising ahuman IgG Fc polypeptide chain; wherein L1 and L3 are at least 15 aminoacids long and L2 is less than 12 amino acids long, wherein either V1 isa VH region and V2 is a VL region or vice versa, wherein either V3 is aVH region and V4 is a VL region or vice versa, wherein the Bi-Fc bindsto a target cell and an immune effector cell and/or mediates cytolysisof a target cell displaying a target cell protein by an immune effectorcell, wherein the Fc polypeptide chain in the first polypeptide chaincomprises the charge pair substitutions K409E or K409E and K392D orK392E, and the Fc polypeptide chain in the second polypeptide chaincomprises the charge pair substitutions D399K or D399R and D356K orD356R, or wherein the Fc polypeptide chain in the second polypeptidechain comprises the charge pair substitutions K409E or K409E and K392Dor K392E, and the Fc polypeptide chain in the first polypeptide chaincomprises the charge pair substitutions D399K or D399R and D356K orD356R, and wherein the Bi-Fc comprises: a VH region comprising the aminoacid sequence of SEQ ID NO:7 or 29 or a variant thereof comprising nomore than 10 amino acid substitutions, insertions, and/or deletions of asingle amino acid per 100 amino acids relative to SEQ ID NO:7 or 29, anda VL region comprising the amino acid sequence of SEQ ID NO:8 or 31 or avariant thereof comprising no more than 10 amino acid substitutions,insertions, and/or deletions of a single amino acid per 100 amino acidsrelative to SEQ ID NO:8 or
 31. 24. A method for treating a patienthaving cancer, a fibrotic disease, or a disease mediated by a pathogencomprising administering to the patient a therapeutically effective doseof the Bi-Fc of claim
 1. 25. The method of claim 24, wherein the patienthas cancer and the method further comprises administering radiation, achemotherapeutic agent, or a non-chemotherapeutic, anti-neoplastic agentbefore, after, or concurrently with the administration of the Bi-Fc. 26.A method for treating a patient having cancer, a fibrotic disease, or adisease mediated by a pathogen comprising administering to the patient atherapeutically effective dose of the Bi-Fc of claim
 16. 27. The methodof claim 26, wherein the patient has cancer and the method furthercomprises administering radiation, a chemotherapeutic agent, or anon-chemotherapeutic, anti-neoplastic agent before, after, orconcurrently with the administration of the Bi-Fc.
 28. A pharmaceuticalcomposition comprising a therapeutically effective dose of the Bi-Fc ofclaim 1 and a physiologically acceptable carrier, excipient, and/ordiluent.
 29. A pharmaceutical composition comprising a therapeuticallyeffective dose of the Bi-Fc of claim 16 and a physiologically acceptablecarrier, excipient, and/or diluent.